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1.
Gastroenterology ; 2021 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-34780722

RESUMO

BACKGROUND: Polygenic and environmental factors are underlying causes of inflammatory bowel disease (IBD). We hypothesized that integration of the genetic loci controlling a metabolite's abundance, with known IBD genetic susceptibility loci, may help resolve metabolic drivers of IBD. METHODS: We measured the levels of 1300 metabolites in the serum of 484 ulcerative colitis (UC) and 464 Crohn's disease (CD) patients and 365 controls. Differential metabolite abundance was determined for disease status, subtype, clinical and endoscopic disease activity as well as IBD phenotype including disease behavior, location and extent. To inform on the genetic basis underlying metabolic diversity, we integrated metabolite and genomic data. Genetic colocalization and Mendelian randomization (MR) analyses were performed using known IBD risk loci to explore whether any metabolite was causally associated with IBD. RESULTS: We found 173 genetically controlled metabolites (mQTL, 9 novel) within 63 non-overlapping loci (7 novel). Furthermore, several metabolites significantly associated with IBD disease status and activity as defined by clinical and endoscopic indexes. This constitutes a resource for biomarker discovery and IBD biology insights. Using this resource, we show that a novel mQTL for serum butyrate levels containing ACADS was not supported as causal for IBD; replicate the association of serum omega-6 containing lipids with the fatty acid desaturase 1/2 locus and identify these metabolites as causal for CD through MR; and validate a novel association of serum plasmalogen and TMEM229B, which was predicted causal for CD. CONCLUSION: An exploratory analysis combining genetics and unbiased serum metabolome surveys can reveal novel biomarkers of disease activity and potential mediators of pathology in IBD.

2.
Kidney360 ; 2(9): 1441-1454, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34651140

RESUMO

Background: Proximal tubular (PT) cells are enriched in mitochondria and peroxisomes. Whereas mitochondrial fatty acid oxidation (FAO) plays an important role in kidney function by supporting the high-energy requirements of PT cells, the role of peroxisomal metabolism remains largely unknown. EHHADH, also known as L-bifunctional protein, catalyzes the second and third step of peroxisomal FAO. Methods: We studied kidneys of WT and Ehhadh KO mice on a C57BL/6N background using histology, immunohistochemistry, immunofluorescence, immunoblot, RNA-sequencing, and metabolomics. To assess the role of androgens in the kidney phenotype of Ehhadh KO mice, mice underwent orchiectomy. Results: We observed male-specific kidney hypertrophy and glomerular filtration rate reduction in adult Ehhadh KO mice. Transcriptome analysis unveiled a gene expression signature similar to PT injury in acute kidney injury mouse models. This was further illustrated by the presence of KIM-1 (kidney injury molecule-1), SOX-9, and Ki67-positive cells in the PT of male Ehhadh KO kidneys. Male Ehhadh KO kidneys had metabolite changes consistent with peroxisomal dysfunction as well as an elevation in glycosphingolipid levels. Orchiectomy of Ehhadh KO mice decreased the number of KIM-1 positive cells to WT levels. We revealed a pronounced sexual dimorphism in the expression of peroxisomal FAO proteins in mouse kidney, underlining a role of androgens in the kidney phenotype of Ehhadh KO mice. Conclusions: Our data highlight the importance of EHHADH and peroxisomal metabolism in male kidney physiology and reveal peroxisomal FAO as a sexual dimorphic metabolic pathway in mouse kidneys.

3.
Am J Hum Genet ; 108(11): 2099-2111, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34678161

RESUMO

The integration of genomic data into health systems offers opportunities to identify genomic factors underlying the continuum of rare and common disease. We applied a population-scale haplotype association approach based on identity-by-descent (IBD) in a large multi-ethnic biobank to a spectrum of disease outcomes derived from electronic health records (EHRs) and uncovered a risk locus for liver disease. We used genome sequencing and in silico approaches to fine-map the signal to a non-coding variant (c.2784-12T>C) in the gene ABCB4. In vitro analysis confirmed the variant disrupted splicing of the ABCB4 pre-mRNA. Four of five homozygotes had evidence of advanced liver disease, and there was a significant association with liver disease among heterozygotes, suggesting the variant is linked to increased risk of liver disease in an allele dose-dependent manner. Population-level screening revealed the variant to be at a carrier rate of 1.95% in Puerto Rican individuals, likely as the result of a Puerto Rican founder effect. This work demonstrates that integrating EHR and genomic data at a population scale can facilitate strategies for understanding the continuum of genomic risk for common diseases, particularly in populations underrepresented in genomic medicine.


Assuntos
Atenção à Saúde/organização & administração , Predisposição Genética para Doença , Hepatopatias/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Registros Eletrônicos de Saúde , Haplótipos , Heterozigoto , Hispano-Americanos/genética , Homozigoto , Humanos , Porto Rico
4.
J Inherit Metab Dis ; 44(6): 1419-1433, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34564857

RESUMO

Peroxisomes metabolize a specific subset of fatty acids, which include dicarboxylic fatty acids (DCAs) generated by ω-oxidation. Data obtained in vitro suggest that the peroxisomal transporter ABCD3 (also known as PMP70) mediates the transport of DCAs into the peroxisome, but in vivo evidence to support this role is lacking. In this work, we studied an Abcd3 KO mouse model generated by CRISPR-Cas9 technology using targeted and untargeted metabolomics, histology, immunoblotting, and stable isotope tracing technology. We show that ABCD3 functions in hepatic DCA metabolism and uncover a novel role for this peroxisomal transporter in lipid homeostasis. The Abcd3 KO mouse presents with increased hepatic long-chain DCAs, increased urine medium-chain DCAs, lipodystrophy, enhanced hepatic cholesterol synthesis and decreased hepatic de novo lipogenesis. Moreover, our study suggests that DCAs are metabolized by mitochondrial fatty acid ß-oxidation when ABCD3 is not functional, reflecting the importance of the metabolic compartmentalization and communication between peroxisomes and mitochondria. In summary, this study provides data on the role of the peroxisomal transporter ABCD3 in hepatic lipid homeostasis and DCA metabolism, and the consequences of peroxisomal dysfunction for the liver.

5.
Cell Mol Life Sci ; 78(14): 5631-5646, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34110423

RESUMO

Peroxisomes play an essential role in the ß-oxidation of dicarboxylic acids (DCAs), which are metabolites formed upon ω-oxidation of fatty acids. Genetic evidence linking transporters and enzymes to specific DCA ß-oxidation steps is generally lacking. Moreover, the physiological functions of DCA metabolism remain largely unknown. In this study, we aimed to characterize the DCA ß-oxidation pathway in human cells, and to evaluate the biological role of DCA metabolism using mice deficient in the peroxisomal L-bifunctional protein (Ehhadh KO mice). In vitro experiments using HEK-293 KO cell lines demonstrate that ABCD3 and ACOX1 are essential in DCA ß-oxidation, whereas both the bifunctional proteins (EHHADH and HSD17B4) and the thiolases (ACAA1 and SCPx) have overlapping functions and their contribution may depend on expression level. We also show that medium-chain 3-hydroxydicarboxylic aciduria is a prominent feature of EHHADH deficiency in mice most notably upon inhibition of mitochondrial fatty acid oxidation. Using stable isotope tracing methodology, we confirmed that products of peroxisomal DCA ß-oxidation can be transported to mitochondria for further metabolism. Finally, we show that, in liver, Ehhadh KO mice have increased mRNA and protein expression of cholesterol biosynthesis enzymes with decreased (in females) or similar (in males) rate of cholesterol synthesis. We conclude that EHHADH plays an essential role in the metabolism of medium-chain DCAs and postulate that peroxisomal DCA ß-oxidation is a regulator of hepatic cholesterol biosynthesis.


Assuntos
Colesterol/metabolismo , Ácidos Dicarboxílicos/urina , Erros Inatos do Metabolismo Lipídico/patologia , Hepatopatias/patologia , Mitocôndrias/patologia , Enzima Bifuncional do Peroxissomo/fisiologia , Animais , Feminino , Células HEK293 , Homeostase , Humanos , Erros Inatos do Metabolismo Lipídico/etiologia , Hepatopatias/etiologia , Hepatopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo
6.
Life (Basel) ; 11(5)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946784

RESUMO

The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the tricarboxylic acid (TCA) cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the coupling of 2-oxoglutarate metabolism to gene transcription related to tumor cell proliferation and aging. DHTKD1 is a gene encoding 2-oxoadipate dehydrogenase (E1a), which functions in the L-lysine degradation pathway. The potentially damaging variants in DHTKD1 have been associated to the (neuro) pathogenesis of several diseases. Evidence was obtained for the formation of a hybrid complex between the OGDHc and E1a, suggesting a potential cross talk between the two metabolic pathways and raising fundamental questions about their assembly. Here we reviewed the recent findings and advances in understanding of protein-protein interactions in OGDHc and 2-oxoadipate dehydrogenase complex (OADHc), an understanding that will create a scaffold to help design approaches to mitigate the effects of diseases associated with dysfunction of the TCA cycle or lysine degradation. A combination of biochemical, biophysical and structural approaches such as chemical cross-linking MS and cryo-EM appears particularly promising to provide vital information for the assembly of 2-oxoacid dehydrogenase complexes, their function and regulation.

7.
Mol Genet Metab Rep ; 27: 100749, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33868931

RESUMO

Patients with a disorder of mitochondrial long-chain fatty acid ß-oxidation (FAO) have reduced fasting tolerance and may present with hypoketotic hypoglycemia, hepatomegaly, (cardio)myopathy and rhabdomyolysis. Patients should avoid a catabolic state because it increases reliance on FAO as energy source. It is currently unclear whether weight loss through a reduction of caloric intake is safe in patients with a FAO disorder. We used the long-chain acyl-CoA dehydrogenase knockout (LCAD KO) mouse model to study the impact of dietary restriction (DR) on the plasma metabolite profile and cardiac function. For this, LCAD KO and wild type (WT) mice were subjected to DR (70% of ad libitum chow intake) for 4 weeks and compared to ad libitum chow fed mice. We found that DR had a relatively small impact on the plasma metabolite profile of WT and LCAD KO mice. Echocardiography revealed a small decrease in left ventricular systolic function of LCAD KO mice, which was most noticeable after DR, but there was no evidence of DR-induced cardiac remodeling. Our results suggest that weight loss through DR does not have acute and detrimental consequences in a mouse model for FAO disorders.

8.
Nat Commun ; 12(1): 547, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483510

RESUMO

Elevated plasma cholesterol and type 2 diabetes (T2D) are associated with coronary artery disease (CAD). Individuals treated with cholesterol-lowering statins have increased T2D risk, while individuals with hypercholesterolemia have reduced T2D risk. We explore the relationship between lipid and glucose control by constructing network models from the STARNET study with sequencing data from seven cardiometabolic tissues obtained from CAD patients during coronary artery by-pass grafting surgery. By integrating gene expression, genotype, metabolomic, and clinical data, we identify a glucose and lipid determining (GLD) regulatory network showing inverse relationships with lipid and glucose traits. Master regulators of the GLD network also impact lipid and glucose levels in inverse directions. Experimental inhibition of one of the GLD network master regulators, lanosterol synthase (LSS), in mice confirms the inverse relationships to glucose and lipid levels as predicted by our model and provides mechanistic insights.


Assuntos
Glicemia/metabolismo , Doença da Artéria Coronariana/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Metabolismo dos Lipídeos , Modelos Biológicos , Animais , Colesterol/sangue , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/genética , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único
9.
Mol Genet Metab ; 132(2): 139-145, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33483254

RESUMO

The glutaric acidurias are a group of inborn errors of metabolism with different etiologies. Glutaric aciduria type 3 (GA3) is a biochemical phenotype with uncertain clinical relevance caused by a deficiency of succinyl-CoA:glutarate-CoA transferase (SUGCT). SUGCT catalyzes the succinyl-CoA-dependent conversion of glutaric acid into glutaryl-CoA preventing urinary loss of the organic acid. Here, we describe the presence of a GA3 trait in mice of 129 substrains due to SUGCT deficiency, which was identified by screening of urine organic acid profiles obtained from different inbred mouse strains including 129S2/SvPasCrl. Molecular and biochemical analyses in an F2 population of the parental C57BL/6J and 129S2/SvPasCrl strains (B6129F2) confirmed that the GA3 trait occurred in Sugct129/129 animals. We evaluated the impact of SUGCT deficiency on metabolite accumulation in the glutaric aciduria type 1 (GA1) mouse model. We found that GA1 mice with SUGCT deficiency have decreased excretion of urine 3-hydroxyglutaric acid and decreased levels glutarylcarnitine in urine, plasma and kidney. Our work demonstrates that SUGCT contributes to the production of glutaryl-CoA under conditions of low and pathologically high glutaric acid levels. Our work also highlights the notion that unexpected biochemical phenotypes can occur in widely used inbred animal lines.


Assuntos
Aciltransferases/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Doenças Metabólicas/genética , Camundongos Endogâmicos/genética , Oxirredutases/deficiência , Transferases/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Animais , Modelos Animais de Doenças , Glutaratos/metabolismo , Humanos , Lisina/metabolismo , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Camundongos , Oxirredutases/genética , Oxirredutases/metabolismo , Fenótipo
10.
Gastroenterology ; 160(1): 287-301.e20, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32980345

RESUMO

BACKGROUND AND AIMS: The presence of gastrointestinal symptoms and high levels of viral RNA in the stool suggest active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication within enterocytes. METHODS: Here, in multiple, large cohorts of patients with inflammatory bowel disease (IBD), we have studied the intersections between Coronavirus Disease 2019 (COVID-19), intestinal inflammation, and IBD treatment. RESULTS: A striking expression of ACE2 on the small bowel enterocyte brush border supports intestinal infectivity by SARS-CoV-2. Commonly used IBD medications, both biologic and nonbiologic, do not significantly impact ACE2 and TMPRSS2 receptor expression in the uninflamed intestines. In addition, we have defined molecular responses to COVID-19 infection that are also enriched in IBD, pointing to shared molecular networks between COVID-19 and IBD. CONCLUSIONS: These data generate a novel appreciation of the confluence of COVID-19- and IBD-associated inflammation and provide mechanistic insights supporting further investigation of specific IBD drugs in the treatment of COVID-19. Preprint doi: https://doi.org/10.1101/2020.05.21.109124.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/enzimologia , Doenças Inflamatórias Intestinais/enzimologia , Mucosa Intestinal/enzimologia , SARS-CoV-2/patogenicidade , Serina Endopeptidases/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Animais , Anti-Inflamatórios/uso terapêutico , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/genética , COVID-19/virologia , Estudos de Casos e Controles , Ensaios Clínicos como Assunto , Estudos Transversais , Modelos Animais de Doenças , Feminino , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/genética , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/virologia , Estudos Longitudinais , Masculino , Camundongos , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/genética , Transdução de Sinais
11.
Front Immunol ; 11: 591815, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33362773

RESUMO

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 13C-glucose or 0.4 mM 13C-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.


Assuntos
Metabolismo dos Carboidratos , Deleção de Genes , Glucose/metabolismo , Proteínas Mitocondriais/genética , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Animais , Biomarcadores , Citocinas/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/patologia , Oxirredução , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-akt/metabolismo
12.
Front Cardiovasc Med ; 7: 592233, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33344518

RESUMO

Aims/hypothesis: 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 (db/db) mouse hearts. In addition, we explore whether direct effects on glucose metabolism are nullified in the presence of an NHE-1 inhibitor. Methods: Langendorff-perfused type II diabetic db/db mouse hearts were examined in three different series: 1: 13C glucose perfusions (n = 32); 2: 13C palmitate perfusions (n = 13); and 3: 13C glucose + 10 µM Cariporide (specific NHE-1 inhibitor) perfusions (n = 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 (n = 8 per group). Results: 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 13C glucose perfusions (13C labeling of lactate: 58 ± 2% vs. 50 ± 3%, for vehicle and EMPA, respectively; P = 0.02), without changes in other glucose metabolic pathways. In contrast, EMPA increased cardiac labeling in α-ketoglutarate derived from 13C palmitate perfusions (13C labeling of α-KG: 79 ± 1% vs. 86 ± 1% for vehicle and EMPA, respectively; P = 0.01). Inhibition of the NHE by Cariporide abolished EMPA effects on lactate labeling from 13C glucose. Conclusions: 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.

13.
Mol Genet Metab ; 131(1-2): 14-22, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32768327

RESUMO

Lysine degradation via formation of saccharopine is a pathway confined to the mitochondria. The second pathway for lysine degradation, the pipecolic acid pathway, is not yet fully elucidated and known enzymes are localized in the mitochondria, cytosol and peroxisome. The tissue-specific roles of these two pathways are still under investigation. The lysine degradation pathway is clinically relevant due to the occurrence of two severe neurometabolic disorders, pyridoxine-dependent epilepsy (PDE) and glutaric aciduria type 1 (GA1). The existence of three other disorders affecting lysine degradation without apparent clinical consequences opens up the possibility to find alternative therapeutic strategies for PDE and GA1 through pathway modulation. A better understanding of the mechanisms, compartmentalization and interplay between the different enzymes and metabolites involved in lysine degradation is of utmost importance.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Encefalopatias Metabólicas/genética , Epilepsia/genética , Glutaril-CoA Desidrogenase/deficiência , Lisina/metabolismo , Mitocôndrias/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Encefalopatias Metabólicas/metabolismo , Encefalopatias Metabólicas/patologia , Citosol/metabolismo , Epilepsia/metabolismo , Epilepsia/patologia , Glutaril-CoA Desidrogenase/genética , Glutaril-CoA Desidrogenase/metabolismo , Humanos , Lisina/análogos & derivados , Lisina/biossíntese , Redes e Vias Metabólicas/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Especificidade de Órgãos/genética , Peroxissomos/genética , Peroxissomos/metabolismo
14.
ACS Chem Biol ; 15(8): 2041-2047, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32633484

RESUMO

DHTKD1 is the E1 component of the 2-oxoadipate dehydrogenase complex, which is an enzyme involved in the catabolism of (hydroxy-)lysine and tryptophan. Mutations in DHTKD1 have been associated with 2-aminoadipic and 2-oxoadipic aciduria, Charcot-Marie-Tooth disease type 2Q and eosinophilic esophagitis, but the pathophysiology of these clinically distinct disorders remains elusive. Here, we report the identification of adipoylphosphonic acid and tenatoprazole as DHTKD1 inhibitors using targeted and high throughput screening, respectively. We furthermore elucidate the DHTKD1 crystal structure with thiamin diphosphate bound at 2.25 Å. We also report the impact of 10 disease-associated missense mutations on DHTKD1. Whereas the majority of the DHTKD1 variants displayed impaired folding or reduced thermal stability in combination with absent or reduced enzyme activity, three variants showed no abnormalities. Our work provides chemical and structural tools for further understanding of the function of DHTKD1 and its role in several human pathologies.


Assuntos
Complexo Cetoglutarato Desidrogenase/antagonistas & inibidores , Tiamina Pirofosfato/química , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/genética , Estrutura Molecular , Mutação de Sentido Incorreto
15.
J Inherit Metab Dis ; 43(6): 1154-1164, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32567100

RESUMO

Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by acute encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. We investigated the efficacy of substrate reduction through inhibition of 2-aminoadipic semialdehyde synthase (AASS), an enzyme upstream of the defective glutaryl-CoA dehydrogenase (GCDH), in a cell line and mouse model of GA1. We show that loss of AASS function in GCDH-deficient HEK-293 cells leads to an approximately fivefold reduction in the established GA1 clinical biomarker glutarylcarnitine. In the GA1 mouse model, deletion of Aass leads to a 4.3-, 3.8-, and 3.2-fold decrease in the glutaric acid levels in urine, brain, and liver, respectively. Parallel decreases were observed in urine and brain 3-hydroxyglutaric acid levels, and plasma, urine, and brain glutarylcarnitine levels. These in vivo data demonstrate that the saccharopine pathway is the main source of glutaric acid production in the brain and periphery of a mouse model for GA1, and support the notion that pharmacological inhibition of AASS may represent an attractive strategy to treat GA1.


Assuntos
Ácido 2-Aminoadípico/análogos & derivados , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Encefalopatias Metabólicas/metabolismo , Encéfalo/metabolismo , Glutaratos/metabolismo , Glutaril-CoA Desidrogenase/deficiência , Fígado/metabolismo , Ácido 2-Aminoadípico/genética , Ácido 2-Aminoadípico/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Animais , Encéfalo/patologia , Encefalopatias Metabólicas/genética , Encefalopatias Metabólicas/terapia , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Feminino , Glutaril-CoA Desidrogenase/genética , Glutaril-CoA Desidrogenase/metabolismo , Células HEK293 , Humanos , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout
16.
Hum Mol Genet ; 29(7): 1168-1179, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32160276

RESUMO

Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by a specific encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. Substrate reduction through inhibition of DHTKD1, an enzyme upstream of the defective glutaryl-CoA dehydrogenase, has been investigated as a potential therapy, but revealed the existence of an alternative enzymatic source of glutaryl-CoA. Here, we show that loss of DHTKD1 in glutaryl-CoA dehydrogenase-deficient HEK-293 cells leads to a 2-fold decrease in the established GA1 clinical biomarker glutarylcarnitine and demonstrate that oxoglutarate dehydrogenase (OGDH) is responsible for this remaining glutarylcarnitine production. We furthermore show that DHTKD1 interacts with OGDH, dihydrolipoyl succinyltransferase and dihydrolipoamide dehydrogenase to form a hybrid 2-oxoglutaric and 2-oxoadipic acid dehydrogenase complex. In summary, 2-oxoadipic acid is a substrate for DHTKD1, but also for OGDH in a cell model system. The classical 2-oxoglutaric dehydrogenase complex can exist as a previously undiscovered hybrid containing DHTKD1 displaying improved kinetics towards 2-oxoadipic acid.


Assuntos
Acil Coenzima A/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Encefalopatias Metabólicas/genética , Glutaril-CoA Desidrogenase/deficiência , Complexo Cetoglutarato Desidrogenase/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Encefalopatias Metabólicas/metabolismo , Encefalopatias Metabólicas/patologia , Células Cultivadas , Glutaril-CoA Desidrogenase/genética , Glutaril-CoA Desidrogenase/metabolismo , Células HEK293 , Humanos , Cetona Oxirredutases/genética , Especificidade por Substrato/genética
17.
Biochim Biophys Acta Mol Basis Dis ; 1866(5): 165720, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32057943

RESUMO

Carnitine plays an essential role in mitochondrial fatty acid ß-oxidation as a part of a cycle that transfers long-chain fatty acids across the mitochondrial membrane and involves two carnitine palmitoyltransferases (CPT1 and CPT2). Two distinct carnitine acyltransferases, carnitine octanoyltransferase (COT) and carnitine acetyltransferase (CAT), are peroxisomal enzymes, which indicates that carnitine is not only important for mitochondrial, but also for peroxisomal metabolism. It has been demonstrated that after peroxisomal metabolism, specific intermediates can be exported as acylcarnitines for subsequent and final mitochondrial metabolism. There is also evidence that peroxisomes are able to degrade fatty acids that are typically handled by mitochondria possibly after transport as acylcarnitines. Here we review the biochemistry and physiological functions of metabolite exchange between peroxisomes and mitochondria with a special focus on acylcarnitines.


Assuntos
Carnitina Aciltransferases/metabolismo , Carnitina/análogos & derivados , Ácidos Graxos/metabolismo , Mitocôndrias/enzimologia , Peroxissomos/enzimologia , Carnitina/metabolismo
18.
J Inherit Metab Dis ; 43(3): 486-495, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31845336

RESUMO

Secondary carnitine deficiency is commonly observed in inherited metabolic diseases characterised by the accumulation of acylcarnitines such as mitochondrial fatty acid oxidation (FAO) disorders. It is currently unclear if carnitine deficiency and/or acylcarnitine accumulation play a role in the pathophysiology of FAO disorders. The long-chain acyl-CoA dehydrogenase (LCAD) KO mouse is a model for long-chain FAO disorders and is characterised by decreased levels of tissue and plasma free carnitine. Tissue levels of carnitine are controlled by SLC22A5, the plasmalemmal carnitine transporter. Here, we have further decreased carnitine availability in the LCAD KO mouse through a genetic intervention by introducing one defective Slc22a5 allele (jvs). Slc22a5 haploinsufficiency decreased free carnitine levels in liver, kidney, and heart of LCAD KO animals. The resulting decrease in the tissue long-chain acylcarnitines levels had a similar magnitude as the decrease in free carnitine. Levels of cardiac deoxycarnitine, a carnitine biosynthesis intermediate, were elevated due to Slc22a5 haploinsufficiency in LCAD KO mice. A similar increase in heart and muscle deoxycarnitine was observed in an independent experiment using Slc22a5jvs/jvs mice. Cardiac hypertrophy, fasting-induced hypoglycemia and increased liver weight, the major phenotypes of the LCAD KO mouse, were not affected by Slc22a5 haploinsufficiency. This may suggest that secondary carnitine deficiency does not play a major role in the pathophysiology of these phenotypes. Similarly, our data do not support a major role for toxicity of long-chain acylcarnitines in the phenotype of the LCAD KO mouse.


Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/genética , Carnitina/análogos & derivados , Metabolismo dos Lipídeos/efeitos dos fármacos , Miocárdio/metabolismo , Membro 5 da Família 22 de Carreadores de Soluto/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Animais , Cardiomiopatias , Carnitina/deficiência , Carnitina/farmacologia , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Feminino , Haploinsuficiência , Hiperamonemia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Doenças Musculares , Fenótipo , Membro 5 da Família 22 de Carreadores de Soluto/genética
19.
Biochim Biophys Acta Mol Basis Dis ; 1865(10): 2774-2787, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31207289

RESUMO

Zellweger spectrum disorders (ZSDs) are autosomal recessive diseases caused by defective peroxisome assembly. They constitute a clinical continuum from severe early lethal to relatively milder presentations in adulthood. Liver disease is a prevalent symptom in ZSD patients. The underlying pathogenesis for the liver disease, however, is not fully understood. We report a hypomorphic ZSD mouse model, which is homozygous for Pex1-c.2531G>A (p.G844D), the equivalent of the most common pathogenic variant found in ZSD, and which predominantly presents with liver disease. After introducing the Pex1-G844D allele by knock-in, we characterized homozygous Pex1-G844D mice for survival, biochemical parameters, including peroxisomal and mitochondrial functions, organ histology, and developmental parameters. The first 20 post-natal days (P20) were critical for survival of homozygous Pex1-G844D mice (~20% survival rate). Lethality was likely due to a combination of cholestatic liver problems, liver dysfunction and caloric deficit, probably as a consequence of defective bile acid biosynthesis. Survival beyond P20 was nearly 100%, but surviving mice showed a marked delay in growth. Surviving mice showed similar hepatic problems as described for mild ZSD patients, including hepatomegaly, bile duct proliferation, liver fibrosis and mitochondrial alterations. Biochemical analyses of various tissues showed the absence of functional peroxisomes accompanied with aberrant levels of peroxisomal metabolites predominantly in the liver, while other tissues were relatively spared. ur findings show that homozygous Pex1-G844D mice have a predominant liver disease phenotype, mimicking the hepatic pathology of ZSD patients, and thus constitute a good model to study pathogenesis and treatment of liver disease in ZSD patients.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Hepatopatias/etiologia , Hepatopatias/metabolismo , Fígado/metabolismo , Síndrome de Zellweger/complicações , ATPases Associadas a Diversas Atividades Celulares/genética , Alelos , Animais , Modelos Animais de Doenças , Feminino , Fibroblastos , Humanos , Fígado/patologia , Hepatopatias/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Peroxissomos , Fenótipo
20.
Mol Genet Metab ; 126(4): 388-396, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30709776

RESUMO

Inbred mouse strains are a cornerstone of translational research but paradoxically many strains carry mild inborn errors of metabolism. For example, α-aminoadipic acidemia and branched-chain ketoacid dehydrogenase deficiency are known in C57BL/6J mice. Using RNA sequencing, we now reveal the causal variants in Dhtkd1 and Bckdhb, and the molecular mechanism underlying these metabolic defects. C57BL/6J mice have decreased Dhtkd1 mRNA expression due to a solitary long terminal repeat (LTR) in intron 4 of Dhtkd1. This LTR harbors an alternate splice donor site leading to a partial splicing defect and as a consequence decreased total and functional Dhtkd1 mRNA, decreased DHTKD1 protein and α-aminoadipic acidemia. Similarly, C57BL/6J mice have decreased Bckdhb mRNA expression due to an LTR retrotransposon in intron 1 of Bckdhb. This transposable element encodes an alternative exon 1 causing aberrant splicing, decreased total and functional Bckdhb mRNA and decreased BCKDHB protein. Using a targeted metabolomics screen, we also reveal elevated plasma C5-carnitine in 129 substrains. This biochemical phenotype resembles isovaleric acidemia and is caused by an exonic splice mutation in Ivd leading to partial skipping of exon 10 and IVD protein deficiency. In summary, this study identifies three causal variants underlying mild inborn errors of metabolism in commonly used inbred mouse strains.


Assuntos
Erros Inatos do Metabolismo/genética , Camundongos Endogâmicos/genética , Animais , Elementos de DNA Transponíveis/genética , Cetona Oxirredutases/genética , Masculino , Erros Inatos do Metabolismo/diagnóstico , Metabolômica , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fenótipo , Análise de Sequência de RNA
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