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1.
Brain ; 142(11): 3382-3397, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31637422

RESUMO

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.

2.
Mol Nutr Food Res ; 63(19): e1900385, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31327168

RESUMO

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.

3.
N Engl J Med ; 380(15): 1433-1441, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30970188

RESUMO

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Ataxia/genética , Deficiências do Desenvolvimento/genética , Glutaminase/deficiência , Glutaminase/genética , Glutamina/metabolismo , Repetições de Microssatélites , Mutação , Atrofia/genética , Cerebelo/patologia , Pré-Escolar , Feminino , Genótipo , Glutamina/análise , Humanos , Masculino , Fenótipo , Reação em Cadeia da Polimerase , Sequenciamento Completo do Genoma
4.
Orphanet J Rare Dis ; 14(1): 30, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30732635

RESUMO

BACKGROUND: Over 80% of women with X-linked adrenoleukodystrophy (ALD) develop spinal cord disease in adulthood for which treatment is supportive only. For future clinical trials quantitative data on disease progression rates are essential. Moreover, diagnosis can be challenging in ALD women, as the most important diagnostic biomarker is normal in 15-20%. Better biomarkers are needed. The purpose of this single centre cross-sectional follow-up study in women with ALD was to assess whether Expanded Disability Status Scale (EDSS), AMC Linear Disability Scale (ALDS) and Short Form (36) Health Survey (SF-36) can detect disease progression and to model the effect of age and duration of symptoms on the rate of progression. Moreover, we performed a pilot study to assess if a semi-targeted lipidomics approach can identify possible new diagnostic biomarkers. RESULTS: In this study 46 women (baseline clinical data published by our group previously) were invited for a follow-up visit. Newly identified women at our center were also recruited. We analysed 65 baseline and 34 follow-up assessments. Median time between baseline and follow-up was 7.8 years (range 6.4-8.7). Mean age at baseline was 49.2 ± 14.2 years, at follow-up 55.4 ± 10.1. EDSS increased significantly (+ 0.08 points/year), but the other outcome measures did not. Increasing age and duration of symptoms were associated with more disability. For the pilot study we analysed plasma of 20 ALD women and 10 controls with ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry, which identified 100 potential biomarker ratios with strong differentiating properties and non-overlapping data distributions between ALD women and controls. CONCLUSIONS: Progression of spinal cord disease can be detected with EDSS, but not with ALDS or SF-36 after a follow-up period of almost 8 years. Moreover, age and the duration of symptoms seem positively associated with the rate of progression. Although a significant progression was measurable, it was below the rate generally conceived as clinically relevant. Therefore, EDSS, ALDS and SF-36 are not suitable as primary outcome measures in clinical trials for spinal cord disease in ALD women. In addition, a semi-targeted lipidomics approach can identify possible new diagnostic biomarkers for women with ALD.


Assuntos
Adrenoleucodistrofia/patologia , Adrenoleucodistrofia/sangue , Adulto , Biomarcadores/sangue , Biologia Computacional , Estudos Transversais , Progressão da Doença , Feminino , Humanos , Modelos Logísticos , Pessoa de Meia-Idade , Doenças da Medula Espinal/patologia
5.
Front Mol Biosci ; 5: 96, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30488036

RESUMO

Metabolism is one of the attributes of life and supplies energy and building blocks to organisms. Therefore, understanding metabolism is crucial for the understanding of complex biological phenomena. Despite having been in the focus of research for centuries, our picture of metabolism is still incomplete. Metabolomics, the systematic analysis of all small molecules in a biological system, aims to close this gap. In order to facilitate such investigations a blueprint of the metabolic network is required. Recently, several metabolic network reconstructions for the model organism Caenorhabditis elegans have been published, each having unique features. We have established the WormJam Community to merge and reconcile these (and other unpublished models) into a single consensus metabolic reconstruction. In a series of workshops and annotation seminars this model was refined with manual correction of incorrect assignments, metabolite structure and identifier curation as well as addition of new pathways. The WormJam consensus metabolic reconstruction represents a rich data source not only for in silico network-based approaches like flux balance analysis, but also for metabolomics, as it includes a database of metabolites present in C. elegans, which can be used for annotation. Here we present the process of model merging, correction and curation and give a detailed overview of the model. In the future it is intended to expand the model toward different tissues and put special emphasizes on lipid metabolism and secondary metabolism including ascaroside metabolism in accordance to their central role in C. elegans physiology.

6.
Cell Rep ; 25(8): 2044-2052.e5, 2018 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-30463003

RESUMO

Metabolic reprogramming has emerged as a crucial regulator of immune cell activation, but how systemic metabolism influences immune cell metabolism and function remains to be investigated. To investigate the effect of dyslipidemia on immune cell metabolism, we performed in-depth transcriptional, metabolic, and functional characterization of macrophages isolated from hypercholesterolemic mice. Systemic metabolic changes in such mice alter cellular macrophage metabolism and attenuate inflammatory macrophage responses. In addition to diminished maximal mitochondrial respiration, hypercholesterolemia reduces the LPS-mediated induction of the pentose phosphate pathway (PPP) and the Nrf2-mediated oxidative stress response. Our observation that suppression of the PPP diminishes LPS-induced cytokine secretion supports the notion that this pathway contributes to inflammatory macrophage responses. Overall, this study reveals that systemic and cellular metabolism are strongly interconnected, together dictating macrophage phenotype and function.

7.
Exp Gerontol ; 113: 128-140, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30300667

RESUMO

Impaired insulin/IGF-1 signaling (IIS) and caloric restriction (CR) prolong lifespan in the nematode C. elegans. However, a cross comparison of these longevity pathways using a multi-omics integration approach is lacking. In this study, we aimed to identify key pathways and metabolite fingerprints of longevity that are shared between IIS and CR worm models using multi-omics integration. We generated transcriptomics and metabolomics data from long-lived worm strains, i.e. daf-2 (impaired IIS) and eat-2 (CR model) and compared them with the wild-type strain N2. Transcriptional profiling identified shared longevity signatures, such as an upregulation of lipid storage and defense responses, and downregulation of macromolecule synthesis and developmental processes. Metabolomics profiling identified an increase in the levels of glycerol­3P, adenine, xanthine, and AMP, and a decrease in the levels of the amino acid pool, as well as the C18:0, C17:1, C19:1, C20:0 and C22:0 fatty acids. After we integrated transcriptomics and metabolomics data based on the annotations in KEGG, our results highlighted increased amino acid metabolism and an upregulation of purine metabolism as a commonality between the two long-lived mutants. Overall, our findings point towards the existence of shared metabolic pathways that are likely important for lifespan extension and provide novel insights into potential regulators and metabolic fingerprints for longevity.

8.
Biochim Biophys Acta Mol Basis Dis ; 1864(11): 3650-3658, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30251684

RESUMO

Barth syndrome (BTHS) is a rare X-linked disorder that is characterized by cardiac and skeletal myopathy, neutropenia and growth abnormalities. The disease is caused by mutations in the tafazzin (TAZ) gene encoding an enzyme involved in the acyl chain remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, this leads to decreased levels of mature CL and accumulation of the intermediate monolysocardiolipin (MLCL). At a cellular level, this causes mitochondrial fragmentation and reduced stability of the respiratory chain supercomplexes. However, the exact mechanism through which tafazzin deficiency leads to disease development remains unclear. We therefore aimed to elucidate the pathways affected in BTHS cells by employing proteomic and metabolic profiling assays. Complexome profiling of patient skin fibroblasts revealed significant effects for about 200 different mitochondrial proteins. Prominently, we found a specific destabilization of higher order oxidative phosphorylation (OXPHOS) supercomplexes, as well as changes in complexes involved in cristae organization and CL trafficking. Moreover, the key metabolic complexes 2-oxoglutarate dehydrogenase (OGDH) and branched-chain ketoacid dehydrogenase (BCKD) were profoundly destabilized in BTHS patient samples. Surprisingly, metabolic flux distribution assays using stable isotope tracer-based metabolomics did not show reduced flux through the TCA cycle. Overall, insights from analyzing the impact of TAZ mutations on the mitochondrial complexome provided a better understanding of the resulting functional and structural consequences and thus the pathological mechanisms leading to Barth syndrome.

9.
Orphanet J Rare Dis ; 13(1): 146, 2018 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-30143026

RESUMO

BACKGROUND: Clinical outcome of patients with a classical presentation of galactosemia (classical patients) varies substantially, even between patients with the same genotype. With current biomarkers, it is not possible to predict clinical outcome early in life. The aim of this study was to develop a method to provide more insight into galactose metabolism, which allows quantitative assessment of residual galactose metabolism in galactosemia patients. We therefore developed a method for galactose metabolite profiling (GMP) in fibroblasts using [U-13C]-labeled galactose. METHODS: GMP analysis was performed in fibroblasts of three classical patients, three variant patients and three healthy controls. The following metabolites were analyzed: [U13C]-galactose, [U13C]-galactose-1-phosphate (Gal-1-P) and [13C6]- uridine diphosphate(UDP)-galactose. The ratio of [U13C]-Gal-1-P/ [13C6]-UDP-galactose was defined as the galactose index (GI). RESULTS: All patient cell lines could be distinguished from the control cell lines and there was a clear difference between variant and classical patients. Variant patients had lower levels of [U13C]-galactose and [U13C]-Gal-1-P than classical patients (though substantially higher than healthy controls) and higher levels of [13C6]-UDP-galactose than classical patients (though substantially lower than healthy controls) resulting in a different GI in all groups. CONCLUSIONS: GMP in fibroblasts is a sensitive method to determine residual galactose metabolism capacity, which can discriminate between patients with a classical presentation of galactosemia, patients with a variant presentation and healthy controls. GMP may be a useful method for early prognostication after further validation in a larger cohort of patients representing the full phenotypic spectrum of galactosemia.

10.
Cell Rep ; 24(8): 2127-2140.e7, 2018 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-30134173

RESUMO

Activation of macrophages by inflammatory stimuli induces reprogramming of mitochondrial metabolism to support the production of pro-inflammatory cytokines and nitric oxide. Hallmarks of this metabolic rewiring are downregulation of α-ketoglutarate formation by isocitrate dehydrogenase (IDH) and accumulation of glutamine-derived succinate, which enhances the inflammatory response via the activity of succinate dehydrogenase (SDH). Here, we identify the nuclear receptor Nur77 (Nr4a1) as a key upstream transcriptional regulator of this pro-inflammatory metabolic switch in macrophages. Nur77-deficient macrophages fail to downregulate IDH expression and accumulate higher levels of succinate and other TCA cycle-derived metabolites in response to inflammatory stimulation in a glutamine-independent manner. Consequently, these macrophages produce more nitric oxide and pro-inflammatory cytokines in an SDH-dependent manner. In vivo, bone marrow Nur77 deficiency exacerbates atherosclerosis development and leads to increased circulating succinate levels. In summary, Nur77 induces an anti-inflammatory metabolic state in macrophages that protects against chronic inflammatory diseases such as atherosclerosis.

11.
Sci Rep ; 8(1): 9562, 2018 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-29934543

RESUMO

Activation of brown adipose tissue (BAT) contributes to total body energy expenditure through energy dissipation as heat. Activated BAT increases the clearance of lipids and glucose from the circulation, but how BAT accommodates large influx of multiple substrates is not well defined. The purpose of this work was to assess the metabolic fluxes in brown adipocytes during ß3-adrenergic receptor (ß3-AR) activation.T37i murine preadipocytes were differentiated into brown adipocytes and we used Seahorse respirometry employing a set of specific substrate inhibitors in the presence or absence of ß3-AR agonist CL316,243. The main substrate used by these brown adipocytes were fatty acids, which were oxidized equally during activation as well as during resting condition. [U-13C]-glucose tracer-based metabolomics revealed that the flux through the TCA cycle was enhanced and regulated by pyruvate dehydrogenase (PDH) activity. Based on 13C-tracer incorporation in lipids, it appeared that most glucose was oxidized via TCA cycle activity, while some was utilized for glycerol-3-phosphate synthesis to replenish the triglyceride pool. Collectively, we show that while fatty acids are the main substrates for oxidation, glucose is also oxidized to meet the increased energy demand during short term ß3-AR activation. PDH plays an important role in directing glucose carbons towards oxidation.

12.
Cardiovasc Res ; 114(10): 1324-1334, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29635338

RESUMO

Aims: Mitochondrial fatty acid oxidation (FAO) is an important energy provider for cardiac work and changes in cardiac substrate preference are associated with different heart diseases. Carnitine palmitoyltransferase 1B (CPT1B) is thought to perform the rate limiting enzyme step in FAO and is inhibited by malonyl-CoA. The role of CPT1B in cardiac metabolism has been addressed by inhibiting or decreasing CPT1B protein or after modulation of tissue malonyl-CoA metabolism. We assessed the role of CPT1B malonyl-CoA sensitivity in cardiac metabolism. Methods and results: We generated and characterized a knock in mouse model expressing the CPT1BE3A mutant enzyme, which has reduced sensitivity to malonyl-CoA. In isolated perfused hearts, FAO was 1.9-fold higher in Cpt1bE3A/E3A hearts compared with Cpt1bWT/WT hearts. Metabolomic, proteomic and transcriptomic analysis showed increased levels of malonylcarnitine, decreased concentration of CPT1B protein and a small but coordinated downregulation of the mRNA expression of genes involved in FAO in Cpt1bE3A/E3A hearts, all of which aim to limit FAO. In vivo assessment of cardiac function revealed only minor changes, cardiac hypertrophy was absent and histological analysis did not reveal fibrosis. Conclusions: Malonyl-CoA-dependent inhibition of CPT1B plays a crucial role in regulating FAO rate in the heart. Chronic elevation of FAO has a relatively subtle impact on cardiac function at least under baseline conditions.

13.
Anal Bioanal Chem ; 410(10): 2551-2568, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29497765

RESUMO

Oxidative stress and inflammation are underlying pathogenic mechanisms associated with the progression of several pathological conditions and immunological responses. Elucidating the role of signalling lipid classes, which include, among others, the isoprostanes, nitro fatty acids, prostanoids, sphingoid bases and lysophosphatidic acids, will create a snapshot of the cause and effect of inflammation and oxidative stress at the metabolic level. Here we describe a fast, sensitive, and targeted ultra-high-performance liquid chromatography-tandem mass spectrometry metabolomics method that allows the quantitative measurement and biological elucidation of 17 isoprostanes as well as their respective isomeric prostanoid mediators, three nitro fatty acids, four sphingoid mediators, and 24 lysophosphatidic acid species from serum as well as organ tissues, including liver, lung, heart, spleen, kidney and brain. Application of this method to paired mouse serum and tissue samples revealed tissue- and serum-specific stress and inflammatory readouts. Little correlation was found between localized (tissue) metabolite levels compared with the systemic (serum) circulation in a homeostatic model. The application of this method in future studies will enable us to explore the role of signalling lipids in the metabolic pathogenicity of stress and inflammation during health and disease.


Assuntos
Inflamação/metabolismo , Metaboloma , Metabolômica/métodos , Estresse Nitrosativo , Estresse Oxidativo , Animais , Cromatografia Líquida de Alta Pressão/métodos , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Humanos , Isoprostanos/análise , Isoprostanos/metabolismo , Lisofosfolipídeos/análise , Lisofosfolipídeos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Espectrometria de Massas em Tandem/métodos
14.
Metabolomics ; 14(10): 122, 2018 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-30830420

RESUMO

INTRODUCTION: Most organisms display circadian rhythms in physiology and behaviour. In mammals, these rhythms are orchestrated by the suprachiasmatic nucleus (SCN). Recently, several metabolites have emerged as important regulators of circadian timekeeping. Metabolomics approaches have aided in identifying some key metabolites in circadian processes in peripheral tissue, but methods to routinely measure metabolites in small brain areas are currently lacking. OBJECTIVE: The aim of the study was to establish a reliable method for metabolite quantifications in the central circadian clock and relate them to different states of neuronal excitability. METHODS: We developed a method to collect and process small brain tissue samples (0.2 mm3), suitable for liquid chromatography-mass spectrometry. Metabolites were analysed in the SCN and one of its main hypothalamic targets, the paraventricular nucleus (PVN). Tissue samples were taken at peak (midday) and trough (midnight) of the endogenous rhythm in SCN electrical activity. Additionally, neuronal activity was altered pharmacologically. RESULTS: We found a minor effect of day/night fluctuations in electrical activity or silencing activity during the day. In contrast, increasing electrical activity during the night significantly upregulated many metabolites in SCN and PVN. CONCLUSION: Our method has shown to produce reliable and physiologically relevant metabolite data from small brain samples. Inducing electrical activity at night mimics the effect of a light pulses in the SCN, producing phase shifts of the circadian rhythm. The upregulation of metabolites could have a functional role in this process, since they are not solely products of physiological states, they are significant parts of cellular signalling pathways.


Assuntos
Relógios Circadianos , Metabolômica , Neurônios/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Núcleo Supraquiasmático/metabolismo , Animais , Estimulação Elétrica , Masculino , Camundongos , Camundongos Endogâmicos C57BL
15.
FASEB J ; 28(7): 2891-900, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24648546

RESUMO

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an inherited disorder of mitochondrial long-chain fatty acid ß-oxidation (FAO). Patients with VLCAD deficiency may present with hypoglycemia, hepatomegaly, cardiomyopathy, and myopathy. Although several mouse models have been developed to aid in the study of the pathogenesis of long-chain FAO defects, the muscular phenotype is underexposed. To address the muscular phenotype, we used a newly developed mouse model on a mixed genetic background with a more severe defect in FAO (LCAD(-/-); VLCAD(+/-)) in addition to a validated mouse model (LCAD(-/-); VLCAD(+/+)) and compared them with wild-type (WT) mice. We found that both mouse models show a 20% reduction in energy expenditure (EE) and a 3-fold decrease in locomotor activity in the unfed state. In addition, we found a 1.7°C drop in body temperature in unfed LCAD(-/-); VLCAD(+/+) mice compared with WT body temperature. We conclude that food withdrawal-induced inactivity, hypothermia, and reduction in EE are novel phenotypes associated with FAO deficiency in mice. Unexpectedly, inactivity was not explained by rhabdomyolysis, but rather reflected the overall reduced capacity of these mice to generate heat. We suggest that mice are partly protected against the negative consequence of an FAO defect.-Diekman, E. F., van Weeghel, M., Wanders, R. J. A., Visser, G., Houten, S. M. Food withdrawal lowers energy expenditure and induces inactivity in long-chain fatty acid oxidation-deficient mouse models.


Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Metabolismo Energético/fisiologia , Ácidos Graxos/metabolismo , Erros Inatos do Metabolismo Lipídico/fisiopatologia , Doenças Mitocondriais/fisiopatologia , Doenças Musculares/fisiopatologia , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Animais , Temperatura Corporal/fisiologia , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Modelos Animais de Doenças , Fígado Gorduroso/metabolismo , Fígado Gorduroso/fisiopatologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Erros Inatos do Metabolismo Lipídico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Doenças Mitocondriais/metabolismo , Atividade Motora/fisiologia , Doenças Musculares/metabolismo , Oxirredução , Fenótipo , Rabdomiólise/metabolismo , Rabdomiólise/fisiopatologia
16.
FASEB J ; 28(3): 1365-74, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24344334

RESUMO

Oxidation of unsaturated fatty acids requires the action of auxiliary enzymes, such as Δ(3),Δ(2)-enoyl-CoA isomerases. Here we describe a detailed biochemical, molecular, histological, and evolutionary characterization of Eci3, the fourth member of the mammalian enoyl-CoA isomerase family. Eci3 specifically evolved in rodents after gene duplication of Eci2. Eci3 is with 79% identity homologous to Eci2 and contains a peroxisomal targeting signal type 1. Subcellular fractionation of mouse kidney and immunofluorescence studies revealed a specific peroxisomal localization for Eci3. Expression studies showed that mouse Eci3 is almost exclusively expressed in kidney. By using immunohistochemistry, we found that Eci3 is not only expressed in cells of the proximal tubule, but also in a subset of cells in the tubulointerstitium and the glomerulus. In vitro, Eci3 catalyzed the isomerization of trans-3-nonenoyl-CoA to trans-2-nonenoyl-CoA equally efficient as Eci2, suggesting a role in oxidation of unsaturated fatty acids. However, in contrast to Eci2, in silico gene coexpression and enrichment analysis for Eci3 in kidney did not yield carboxylic acid metabolism, but diverse biological functions, such as ion transport (P=7.1E-3) and tissue morphogenesis (P=1.0E-3). Thus, Eci3 picked up a novel and unexpected role in kidney function during rodent evolution.


Assuntos
Dodecenoil-CoA Isomerase/metabolismo , Rim/enzimologia , Animais , Sequência de Bases , Primers do DNA , Imunofluorescência , Humanos , Camundongos
17.
J Inherit Metab Dis ; 36(6): 973-81, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23563854

RESUMO

PURPOSE: Elevation of long-chain acylcarnitine levels is a hallmark of long-chain mitochondrial ß-oxidation (FAO) disorders, and can be accompanied by secondary carnitine deficiency. To restore free carnitine levels, and to increase myocardial export of long-chain fatty acyl-CoA esters, supplementation of L-carnitine in patients has been proposed. However, carnitine supplementation is controversial, because it may enhance the potentially lipotoxic buildup of long-chain acylcarnitines in the FAO-deficient heart. In this longitudinal study, we investigated the effects of carnitine supplementation in an animal model of long-chain FAO deficiency, the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse. METHODS: Cardiac size and function, and triglyceride (TG) levels were quantified using proton magnetic resonance imaging (MRI) and spectroscopy ((1)H-MRS) in LCAD KO and wild-type (WT) mice. Carnitine was supplemented orally for 4 weeks starting at 5 weeks of age. Non-supplemented animals served as controls. In vivo data were complemented with ex vivo biochemical assays. RESULTS: LCAD KO mice displayed cardiac hypertrophy and elevated levels of myocardial TG compared to WT mice. Carnitine supplementation lowered myocardial TG, normalizing myocardial TG levels in LCAD KO mice. Furthermore, carnitine supplementation did not affect cardiac performance and hypertrophy, or induce an accumulation of potentially toxic long-chain acylcarnitines in the LCAD KO heart. CONCLUSION: This study lends support to the proposed beneficial effect of carnitine supplementation alleviating toxicity by exporting acylcarnitines out of the FAO-deficient myocardium, rather than to the concern about a potentially detrimental effect of supplementation-induced production of lipotoxic long-chain acylcarnitines.


Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/genética , Carnitina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Miocárdio/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Animais , Carnitina/análogos & derivados , Carnitina/sangue , Suplementos Nutricionais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Coração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Triglicerídeos/metabolismo
18.
FASEB J ; 26(10): 4316-26, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22782973

RESUMO

Mitochondrial enoyl-CoA isomerase (ECI1) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild-type (WT) and Eci1-deficient knockout (KO) mice to explore a potential presentation of human ECI1 deficiency. Upon food withdrawal, Eci1-deficient mice displayed normal blood ß-hydroxybutyrate levels (WT 1.09 mM vs. KO 1.10 mM), a trend to lower blood glucose levels (WT 4.58 mM vs. KO 3.87 mM, P=0.09) and elevated blood levels of unsaturated acylcarnitines, in particular C12:1 acylcarnitine (WT 0.03 µM vs. KO 0.09 µM, P<0.01). Feeding an olive oil-rich diet induced an even greater increase in C12:1 acylcarnitine levels (WT 0.01 µM vs. KO 0.04 µM, P<0.01). Overall, the phenotypic presentation of Eci1-deficient mice is mild, possibly caused by the presence of a second enoyl-CoA isomerase (Eci2) in mitochondria. Knockdown of Eci2 in Eci1-deficient fibroblasts caused a more pronounced accumulation of C12:1 acylcarnitine on incubation with unsaturated fatty acids (12-fold, P<0.05). We conclude that Eci2 compensates for Eci1 deficiency explaining the mild phenotype of Eci1-deficient mice. Hypoglycemia and accumulation of C12:1 acylcarnitine might be diagnostic markers to identify ECI1 deficiency in humans.


Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Ácidos Graxos Insaturados/metabolismo , Mitocôndrias/enzimologia , Animais , Glicemia/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/genética , Carnitina/análogos & derivados , Carnitina/sangue , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Dodecenoil-CoA Isomerase , Immunoblotting , Espectrometria de Massas , Camundongos , Camundongos Knockout , Oxirredução , Reação em Cadeia da Polimerase em Tempo Real
19.
Endocrinology ; 152(12): 4641-51, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21990309

RESUMO

Dysregulation of fatty acid oxidation plays a pivotal role in the pathophysiology of obesity and insulin resistance. Medium- and short-chain-3-hydroxyacyl-coenzyme A (CoA) dehydrogenase (SCHAD) (gene name, hadh) catalyze the third reaction of the mitochondrial ß-oxidation cascade, the oxidation of 3-hydroxyacyl-CoA to 3-ketoacyl-CoA, for medium- and short-chain fatty acids. We identified hadh as a putative obesity gene by comparison of two genome-wide scans, a quantitative trait locus analysis previously performed in the polygenic obese New Zealand obese mouse and an earlier described small interfering RNA-mediated mutagenesis in Caenorhabditis elegans. In the present study, we show that mice lacking SCHAD (hadh(-/-)) displayed a lower body weight and a reduced fat mass in comparison with hadh(+/+) mice under high-fat diet conditions, presumably due to an impaired fuel efficiency, the loss of acylcarnitines via the urine, and increased body temperature. Food intake, total energy expenditure, and locomotor activity were not altered in knockout mice. Hadh(-/-) mice exhibited normal fat tolerance at 20 C. However, during cold exposure, knockout mice were unable to clear triglycerides from the plasma and to maintain their normal body temperature, indicating that SCHAD plays an important role in adaptive thermogenesis. Blood glucose concentrations in the fasted and postprandial state were significantly lower in hadh(-/-) mice, whereas insulin levels were elevated. Accordingly, insulin secretion in response to glucose and glucose plus palmitate was elevated in isolated islets of knockout mice. Therefore, our data indicate that SCHAD is involved in thermogenesis, in the maintenance of body weight, and in the regulation of nutrient-stimulated insulin secretion.


Assuntos
Acil-CoA Desidrogenase/fisiologia , Peso Corporal , Butiril-CoA Desidrogenase/fisiologia , Insulina/metabolismo , Termogênese , Animais , Glicemia , Temperatura Baixa , Metabolismo Energético , Camundongos , Camundongos Knockout , Triglicerídeos/sangue
20.
Nucleus ; 2(3): 195-207, 2011 May-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21818413

RESUMO

A-type lamins are a major component of the nuclear lamina. Mutations in the LMNA gene, which encodes the A-type lamins A and C, cause a set of phenotypically diverse diseases collectively called laminopathies. While adult LMNA null mice show various symptoms typically associated with laminopathies, the effect of loss of lamin A/C on early post-natal development is poorly understood. Here we developed a novel LMNA null mouse (LMNA(GT-/-)) based on genetrap technology and analyzed its early post-natal development. We detect LMNA transcripts in heart, the outflow tract, dorsal aorta, liver and somites during early embryonic development. Loss of A-type lamins results in severe growth retardation and developmental defects of the heart, including impaired myocyte hypertrophy, skeletal muscle hypotrophy, decreased amounts of subcutaneous adipose tissue and impaired ex vivo adipogenic differentiation. These defects cause death at 2 to 3 weeks post partum associated with muscle weakness and metabolic complications, but without the occurrence of dilated cardiomyopathy or an obvious progeroid phenotype. Our results indicate that defective early post-natal development critically contributes to the disease phenotypes in adult laminopathies.


Assuntos
Adipogenia/genética , Deleção de Genes , Lamina Tipo A/deficiência , Lamina Tipo A/genética , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Desenvolvimento Muscular/genética , Animais , Desenvolvimento Embrionário/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes Reporter/genética , Hipertrofia/genética , Lamina Tipo A/metabolismo , Masculino , Doenças Metabólicas/genética , Doenças Metabólicas/fisiopatologia , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenótipo , Regiões Promotoras Genéticas/genética , Gordura Subcutânea/metabolismo , Gordura Subcutânea/patologia , Transcriptoma
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