RESUMO
Patients with citrin deficiency during the adaptation/compensation period exhibit diverse clinical features and have characteristic diet of high protein, high fat, and low carbohydrate. Japanese cuisine typically contains high carbohydrate but evaluation of diet of citrin-deficient patients in 2008 showed a low energy intake and a protein:fat:carbohydrate (PFC) ratio of 19:44:37, which indicates low carbohydrate consumption rate. These findings prompted the need for diet intervention to prevent the adult onset of type II citrullinemia (CTLN2). Since the publication of the report about 10 years ago, patients are generally advised to eat what they wish under active dietary consultation and intervention. In this study, citrin-deficient patients and control subjects living in the same household provided answers to a questionnaire, filled-up a maximum 6-day food diary, and supplied physical data and information on medications if any. To study the effects of the current diet, the survey collected data from 62 patients and 45 controls comparing daily intakes of energy, protein, fat, and carbohydrate. Food analysis showed that patient's energy intake was 115% compared to the Japanese standard. The confidence interval of the PFC ratio of patients was 20-22:47-51:28-32, indicating higher protein, higher fat and lower carbohydrate relative to previous reports. The mean PFC ratio of female patients (22:53:25) was significantly different from that of male patients (20:46:34), which may explain the lower frequency of CTLN2 in females. Comparison of the present data to those published 10 years ago, energy, protein, and fat intakes were significantly higher but the amount of carbohydrate consumption remained the same. Regardless of age, most patients (except for adolescents) consumed 100-200 g/day of carbohydrates, which met the estimated average requirement of 100 g/day for healthy individuals. Finally, patients were generally not overweight and some CTLN2 patients were underweight although their energy intake was higher compared with the control subjects. We speculate that high-energy of a low carbohydrate diet under dietary intervention may help citrin-deficient patients attain normal growth and prevent the onset of CTLN2.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Citrulinemia/dietoterapia , Metabolismo Energético/fisiologia , Transportadores de Ânions Orgânicos/genética , Adolescente , Adulto , Proteínas de Ligação ao Cálcio/deficiência , Metabolismo dos Carboidratos/fisiologia , Carboidratos/administração & dosagem , Citrulinemia/epidemiologia , Citrulinemia/metabolismo , Citrulinemia/patologia , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/metabolismo , Ingestão de Alimentos/fisiologia , Feminino , Humanos , Japão/epidemiologia , Masculino , Proteínas de Transporte da Membrana Mitocondrial/genética , Transportadores de Ânions Orgânicos/deficiência , Proteínas/administração & dosagem , Proteínas/metabolismoRESUMO
Can you imagine a disease in which intake of an excess amount of sugars or carbohydrates causes hyperammonemia? It is hard to imagine the intake causing hyperammonemia. AGC2 or citrin deficiency shows their symptoms following sugar/carbohydrates intake excess and this disease is now known as a pan-ethnic disease. AGC2 (aspartate glutamate carrier 2) or citrin is a mitochondrial transporter which transports aspartate (Asp) from mitochondria to cytosol in exchange with glutamate (Glu) and H+. Asp is originally supplied from mitochondria to cytosol where it is necessary for synthesis of proteins, nucleotides, and urea. In cytosol, Asp can be synthesized from oxaloacetate and Glu by cytosolic Asp aminotransferase, but oxaloacetate formation is limited by the amount of NAD+. This means an increase in NADH causes suppression of Asp formation in the cytosol. Metabolism of carbohydrates and other substances which produce cytosolic NADH such as alcohol and glycerol suppress oxaloacetate formation. It is forced under citrin deficiency since citrin is a member of malate/Asp shuttle. In this review, we will describe history of identification of the SLC25A13 gene as the causative gene for adult-onset type II citrullinemia (CTLN2), a type of citrin deficiency, pathophysiology of citrin deficiency together with animal models and possible treatments for citrin deficiency newly developing.
Assuntos
Ácido Aspártico/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Citrulinemia/metabolismo , Ácido Glutâmico/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Animais , Transporte Biológico , Proteínas de Ligação ao Cálcio/genética , Citrulinemia/genética , Citrulinemia/terapia , Predisposição Genética para Doença/genética , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Transportadores de Ânions Orgânicos/genéticaRESUMO
Citrin, encoded by SLC25A13 gene, is an inner mitochondrial transporter that is part of the malate-aspartate shuttle, which regulates the NAD+/NADH ratio between the cytosol and mitochondria. Citrullinemia type II (CTLN-II) is an inherited disorder caused by germline mutations in SLC25A13, manifesting clinically in growth failure that can be alleviated by dietary restriction of carbohydrates. The association of citrin with glycolysis and NAD+/NADH ratio led us to hypothesize that it may play a role in carcinogenesis. Indeed, we find that citrin is upregulated in multiple cancer types and is essential for supplementing NAD+ for glycolysis and NADH for oxidative phosphorylation. Consequently, citrin deficiency associates with autophagy, whereas its overexpression in cancer cells increases energy production and cancer invasion. Furthermore, based on the human deleterious mutations in citrin, we found a potential inhibitor of citrin that restricts cancerous phenotypes in cells. Collectively, our findings suggest that targeting citrin may be of benefit for cancer therapy.
Assuntos
Carcinogênese/genética , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Neoplasias/genética , Carboidratos/genética , Citrulinemia/genética , Citrulinemia/metabolismo , Citosol/metabolismo , Citosol/patologia , Regulação Neoplásica da Expressão Gênica/genética , Mutação em Linhagem Germinativa/genética , Glutamatos/farmacologia , Ácido Glutâmico/análogos & derivados , Ácido Glutâmico/farmacologia , Glicólise/genética , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação Oxidativa/efeitos dos fármacosRESUMO
Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options.
Assuntos
Citrulinemia/tratamento farmacológico , Citrulinemia/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Terapia Genética/métodos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , RNA Mensageiro/uso terapêutico , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Glucosefosfato Desidrogenase/genética , Células HeLa , Células Hep G2 , Humanos , Lipídeos/química , Mutação com Perda de Função , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Nanopartículas/química , Fases de Leitura Aberta/genética , RNA Mensageiro/síntese química , RNA Mensageiro/química , RNA Mensageiro/genética , Transfecção , Resultado do TratamentoRESUMO
BACKGROUND: Argininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate. Mutations in the ASS1 gene cause citrullinemia type I, a rare autosomal recessive disorder characterized by neonatal hyperammonemia, elevated citrulline levels, and early neonatal death. Treatment for this disease is currently restricted to liver transplantation; however, due to limited organ availability, substitute therapies are required. Recently, extracellular vesicles (EVs) have been reported to act as intercellular transporters carrying genetic information responsible for cell reprogramming. In previous studies, we isolated a population of stem cell-like cells known as human liver stem cells (HLSCs) from healthy liver tissue. Moreover, EVs derived from HLSCs were reported to exhibit regenerative effects on the liver parenchyma in models of acute liver injury. The aim of this study was to evaluate whether EVs derived from normal HLSCs restored ASS1 enzymatic activity and urea production in hepatocytes differentiated from HLSCs derived from a patient with type I citrullinemia. METHODS: HLSCs were isolated from the liver of a patient with type I citrullinemia (ASS1-HLSCs) and characterized by fluorescence-activated cell sorting (FACS), immunofluorescence, and DNA sequencing analysis. Furthermore, their differentiation capabilities in vitro were also assessed. Hepatocytes differentiated from ASS1-HLSCs were evaluated by the production of urea and ASS enzymatic activity. EVs derived from normal HLSCs were purified by differential ultracentrifugation followed by floating density gradient. The EV content was analyzed to identify the presence of ASS1 protein, mRNA, and ASS1 gene. In order to obtain ASS1-depleted EVs, a knockdown of the ASS1 gene in HLSCs was performed followed by EV isolation from these cells. RESULTS: Treating ASS1-HLSCs with EVs from HLSCs restored both ASS1 activity and urea production mainly through the transfer of ASS1 enzyme and mRNA. In fact, EVs from ASS1-knockdown HLSCs contained low amounts of ASS1 mRNA and protein, and were unable to restore urea production in hepatocytes differentiated from ASS1-HLSCs. CONCLUSIONS: Collectively, these results suggest that EVs derived from normal HLSCs may compensate the loss of ASS1 enzyme activity in hepatocytes differentiated from ASS1-HLSCs.
Assuntos
Argininossuccinato Sintase , Citrulinemia , Vesículas Extracelulares/metabolismo , Fígado/metabolismo , Células-Tronco/metabolismo , Argininossuccinato Sintase/biossíntese , Argininossuccinato Sintase/genética , Citrulinemia/genética , Citrulinemia/metabolismo , Citrulinemia/terapia , Hepatócitos/metabolismo , Humanos , Ureia/metabolismoRESUMO
Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients.
Assuntos
Citrulinemia/metabolismo , Sacarose Alimentar/administração & dosagem , Etanol/administração & dosagem , Glicerol/administração & dosagem , Fígado/química , Trifosfato de Adenosina/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animais , Antiporters/genética , Modelos Animais de Doenças , Glicerolfosfato Desidrogenase/genética , Glicerofosfatos/metabolismo , Humanos , Camundongos , Camundongos KnockoutRESUMO
Cancer cells hijack and remodel existing metabolic pathways for their benefit. Argininosuccinate synthase (ASS1) is a urea cycle enzyme that is essential in the conversion of nitrogen from ammonia and aspartate to urea. A decrease in nitrogen flux through ASS1 in the liver causes the urea cycle disorder citrullinaemia. In contrast to the well-studied consequences of loss of ASS1 activity on ureagenesis, the purpose of its somatic silencing in multiple cancers is largely unknown. Here we show that decreased activity of ASS1 in cancers supports proliferation by facilitating pyrimidine synthesis via CAD (carbamoyl-phosphate synthase 2, aspartate transcarbamylase, and dihydroorotase complex) activation. Our studies were initiated by delineating the consequences of loss of ASS1 activity in humans with two types of citrullinaemia. We find that in citrullinaemia type I (CTLN I), which is caused by deficiency of ASS1, there is increased pyrimidine synthesis and proliferation compared with citrullinaemia type II (CTLN II), in which there is decreased substrate availability for ASS1 caused by deficiency of the aspartate transporter citrin. Building on these results, we demonstrate that ASS1 deficiency in cancer increases cytosolic aspartate levels, which increases CAD activation by upregulating its substrate availability and by increasing its phosphorylation by S6K1 through the mammalian target of rapamycin (mTOR) pathway. Decreasing CAD activity by blocking citrin, the mTOR signalling, or pyrimidine synthesis decreases proliferation and thus may serve as a therapeutic strategy in multiple cancers where ASS1 is downregulated. Our results demonstrate that ASS1 downregulation is a novel mechanism supporting cancerous proliferation, and they provide a metabolic link between the urea cycle enzymes and pyrimidine synthesis.
Assuntos
Argininossuccinato Sintase/deficiência , Ácido Aspártico/metabolismo , Neoplasias/metabolismo , Pirimidinas/biossíntese , Animais , Argininossuccinato Sintase/metabolismo , Aspartato Carbamoiltransferase/metabolismo , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Citrulinemia/metabolismo , Citosol/metabolismo , Di-Hidro-Orotase/metabolismo , Regulação para Baixo , Ativação Enzimática , Humanos , Masculino , Camundongos , Camundongos SCID , Neoplasias/enzimologia , Neoplasias/patologia , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Fosforilação , Serina-Treonina Quinases TOR/metabolismoRESUMO
Loss of argininosuccinate synthetase (ASS) expression in melanoma makes these tumor cells vulnerable to arginine deprivation. Pegylated arginine deiminase (ADI-PEG20) which degrades arginine to citrulline and ammonia has been used clinically and partial responses and stable disease have been noted with minimal toxicity. In order to improve the therapeutic efficacy of ADI-PEG20, we have combined ADI-PEG20 with a DNA damaging agent, cisplatin. We have shown that the combination of the two drugs together significantly improved the therapeutic efficacy when compared to ADI-PEG20 alone or cisplatin alone in 4 melanoma cell lines, regardless of their BRAF mutation. In-vivo study also exhibited the same effect as in-vitro with no added toxicity to either agent alone. The underlying mechanism is complex, but increased DNA damage upon arginine deprivation due to decreased DNA repair proteins, FANCD2, ATM, and CHK1/2 most likely leads to increased apoptosis. This action is further intensified by increased proapoptotic protein, NOXA, and decreased antiapoptotic proteins, SURVIVIN, BCL2 and XIAP. The autophagic process which protects cells from apoptosis upon ADI-PEG20 treatment also dampens upon cisplatin administration. Thus, the combination of arginine deprivation and cisplatin function in concert to kill tumor cells which do not express ASS without added toxicity to normal cells.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Arginina/metabolismo , Cisplatino/farmacologia , Hidrolases/farmacologia , Melanoma/tratamento farmacológico , Polietilenoglicóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Argininossuccinato Sintase/metabolismo , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Citrulinemia/metabolismo , Citrulinemia/patologia , Feminino , Humanos , Hidrolases/administração & dosagem , Melanoma/enzimologia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Polietilenoglicóis/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Citrin-deficient children and adolescents between adult-onset type II citrullinemia and neonatal intrahepatic cholestasis by citrin deficiency do not have clear clinical features except for unusual diet of high-fat, high-protein, and low-carbohydrate food. The aims of the present study are to characterize fatigue and quality of life (QOL) in citrin-deficient patients during adaptation and compensation stage, and to define the relationship between fatigue and QOL. The study subjects were 55 citrin-deficient patients aged 1-22years (29 males) and 54 guardians. Fatigue was evaluated by self-reports and proxy-reports of the PedsQL Multidimensional Fatigue Scale. QOL was evaluated by the PedsQL Generic Core Scales. Both scale scores were significantly lower in child self-reports (p<0.01 and p<0.05, respectively) and parent proxy-reports (p<0.01 and p<0.01, respectively) than those of healthy children. Citrin-deficient patients with scores of 50 percentile or less of healthy children constituted 67.5% of the sample for the Fatigue Scale and 68.4% for the Generic Core Scales. The PedsQL Fatigue Scale correlated with the Generic Core Scales for both the patients (r=0.56) and parents reports (r=0.71). Assessments by the patients and their parents showed moderate agreement. Parents assessed the condition of children more favorably than their children. The study identified severe fatigue and impaired QOL in citrin-deficient patients during the silent period, and that such children perceive worse fatigue and poorer QOL than those estimated by their parents. The results stress the need for active involvement of parents and medical staff in the management of citrin-deficient patients during the silent period.
Assuntos
Adaptação Fisiológica , Metabolismo dos Carboidratos , Citrulinemia/metabolismo , Citrulinemia/patologia , Fadiga/metabolismo , Adolescente , Proteínas de Ligação ao Cálcio/deficiência , Criança , Pré-Escolar , Citrulinemia/terapia , Dieta Hiperlipídica , Fadiga/patologia , Fadiga/terapia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Transportadores de Ânions Orgânicos/deficiência , Qualidade de Vida , Adulto JovemRESUMO
Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is an autosomal recessive disease caused by the dysfunction of citrin, an aspartate/glutamate carrier encoded by the SLC25A13 gene. Considerable progress has been made on the diagnosis and treatment of NICCD, but its clinical and molecular features still remain far from being completely elucidated and generally understood. The infant, a preterm female delivered at a gestational age of 31 weeks, was referred to our hospital at the age of 8 months because of jaundice lasting for 4.5 months and ovarian masses uncovered for 3 months. Besides serum indices indicating cholestasis, elevated serum levels of luteinizing hormone, follicle stimulating hormone and estradiol were also detected. Abdominal magnetic resonance imaging demonstrated bilateral multi-cystic ovarian masses, with the largest size being 7.4 × 6.2 × 9.6 mm(3). SLC25A13 gene analysis revealed that the patient was a compound heterozygote of c.1177+1G>A and c.754G>A. The paternally-inherited mutation c.754G>A was a novel one with a carrier rate of less than 1%. SLC25A13 transcriptional study in peripheral blood lymphocytes (PBLs) documented a novel splice variant r.616_848del which resulted from c.754G>A, with another variant r.1019_1177del from the maternally-inherited c.1177+1G>A mutation. The diagnoses were NICCD and multiple ovarian antral follicles (minipuberty), and the patient responded well to a galactose-free and medium chain triglyceride (MCT)-enriched formula. The findings in this paper expanded the clinical and molecular spectrum of the SLC25A13 gene, and lent support to the concept that PBLs could be taken as a feasible specimen source for SLC25A13 transcriptional analysis.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Citrulinemia/genética , Citrulinemia/metabolismo , Transportadores de Ânions Orgânicos/genética , Folículo Ovariano/fisiopatologia , Citrulinemia/sangue , Citrulinemia/diagnóstico , Citrulinemia/dietoterapia , Estradiol/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Hormônio Luteinizante/sangue , Linfócitos/metabolismo , Imageamento por Ressonância Magnética , Mutação , Cistos Ovarianos/sangue , Cistos Ovarianos/diagnóstico , Cistos Ovarianos/dietoterapia , Cistos Ovarianos/genética , Cistos Ovarianos/metabolismo , Transcrição Gênica , Resultado do Tratamento , Triglicerídeos/uso terapêuticoRESUMO
OBJECTIVE: To assess the feasibility of high-resolution melting (HRM) analysis for screening patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). METHODS: Based on previous studies on SLC25A13 gene in Chinese patients with NICCD, four hotspot mutations (851del4, 1638ins23, IVS6+5G>A and IVS16ins3kb) were selected. Results of the HRM analysis was validated using 50 negative controls and 20 patients with NICCD whose genotypes were confirmed previously by direct sequencing. With the established protocol, 171 suspected patients were enrolled. Samples with abnormal melting curves were further validated by DNA sequencing. RESULTS: HRM analysis can accurately determine the genotypes of all negative controls and patients. The sensitivity and specificity of the technique reached 100% (70/70). The melting curves of samples with the same genotype were highly reproducible. In 171 suspected patients, seven NICCD patients were detected by HRM. Identified mutations have included one case of 851del4 homozygote, one case of IVS6+5G>A heterozygote, 3 cases of 851del4 heterozygotes, one case of [IVS6+5G>A]+[ 851del4] and one case of [1638ins23+IVS16ins3kb]+[1638ins23]. All mutations were subsequently confirmed by DNA sequencing. CONCLUSION: HRM analysis is a convenient, high-throughput and rapid technique for the screening of NICCD patients.
Assuntos
Proteínas de Ligação ao Cálcio/deficiência , Citrulinemia/diagnóstico , Citrulinemia/genética , DNA/química , Transportadores de Ânions Orgânicos/deficiência , Proteínas de Transporte de Ânions/genética , Sequência de Bases , China , Citrulinemia/metabolismo , DNA/genética , Predisposição Genética para Doença , Genótipo , Humanos , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Mutação , Desnaturação de Ácido Nucleico , Sensibilidade e EspecificidadeRESUMO
BACKGROUND/AIMS: Citrin deficiency caused by SLC25A13 gene mutations develops into adult-onset type II citrullinemia (CTLN2) and may be accompanied with hepatic steatosis and steatohepatitis. As its clinical features remain unclear, we aimed to explore the characteristics of fatty liver disease associated with citrin deficiency. METHODS: The prevalence of hepatic steatosis in 19 CTLN2 patients was examined, and clinical features were compared with those of non-alcoholic fatty liver disease (NAFLD) patients without known SLC25A13 gene mutations. RESULTS: Seventeen (89%) CTLN2 patients had steatosis, and 4 (21%) had been diagnosed as having NAFLD before appearance of neuropsychological symptoms. One patient had steatohepatitis. Citrin deficiency-associated fatty livers showed a considerably lower prevalence of accompanying obesity and metabolic syndrome, higher prevalence of history of pancreatitis, and higher serum levels of pancreatic secretory trypsin inhibitor (PSTI) than fatty livers without the mutations. Receiver operating characteristic curve analyses revealed that a body mass index < 20kg/m(2) and serum PSTI>29ng/mL were associated with citrin deficiency. CONCLUSIONS: Patients presenting with non-alcoholic fatty liver unrelated to obesity and metabolic syndrome might have citrin deficiency, and serum PSTI may be a useful indicator for distinguishing this from conventional NAFLD.
Assuntos
Citrulinemia/etiologia , Fígado Gorduroso/etiologia , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Adulto , Idoso , Proteínas de Transporte/sangue , Citrulinemia/diagnóstico , Citrulinemia/genética , Citrulinemia/metabolismo , Diagnóstico Diferencial , Fígado Gorduroso/diagnóstico , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Feminino , Humanos , Masculino , Síndrome Metabólica/complicações , Pessoa de Meia-Idade , Proteínas de Transporte da Membrana Mitocondrial , Modelos Biológicos , Mutação , Obesidade/complicações , Inibidor da Tripsina Pancreática de Kazal , Adulto JovemRESUMO
Citrin is a mitochondrial aspartate-glutamate carrier primarily expressed in liver. Adult-onset type II citrullinemia is caused by mutations in the SLC25A13 gene that encodes for citrin, and patients with this condition do not express citrin. We found apoptotic hepatocytes in one such patient. This finding prompted us to investigate the role of citrin in hepatocyte survival. Knockdown of citrin by a vector-based short-hairpin RNA technique reduced cell viability and induced apoptosis of a hepatocellular carcinoma cell line, Hep3B cells. Caspase-3/7 and caspase-9 were activated, and PARP was cleaved. Citrin knockdown also increased the expression of Bax and Bak, and reduced expression of Bcl-xL and Bcl-2. These alterations resulted in the release of cytochrome c from the mitochondria. Our results indicated that citrin downregulation induces apoptosis of hepatocytes through the mitochondrial death pathway, highlighting the importance of citrin in survival of hepatocytes and maintenance of liver function.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Antiporters/metabolismo , Apoptose , Proteínas de Ligação ao Cálcio/metabolismo , Regulação para Baixo/genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Mitocôndrias/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Ligação ao Cálcio/genética , Caspases/metabolismo , Células Cultivadas , Citrulinemia/genética , Citrulinemia/metabolismo , Citrulinemia/patologia , Citocromos c/metabolismo , Humanos , Transportadores de Ânions Orgânicos/genética , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND/AIMS: Mutations in SLC25A13, encoding the mitochondrial aspartate-glutamate carrier citrin, cause adult-onset type II citrullinemia (CTLN2) in humans. We have previously reported that although citrin-knockout (Ctrn-/-) mice fail to display symptoms of CTLN2, liver perfusion revealed a deficit in ureogenesis from ammonia accompanied by an increase in the perfusate lactate-to-pyruvate (L/P) ratio. The present study explores the effects of pyruvate, aspartate and citrate on improving the abnormalities observed in the Ctrn-/- liver. METHODS: We measured the rate of ureogenesis from ammonium chloride using the liver-perfusion system. RESULTS: Pyruvate infusion lowered the L/P ratio and corrected the deficit in ureogenesis in the Ctrn-/- liver. This effect was found to be dose-dependent in both instances. Phenazine methosulfate, a cytosolic oxidant, also improved the rate of ureogenesis in the Ctrn-/- liver and led to a fall in the L/P ratio. The addition of aspartate or citrate did not change either the rate of ureogenesis or the L/P ratio in the Ctrn-/- liver. CONCLUSIONS: Citrin deficiency disturbs urea synthesis primarily as a result of an elevated cytosolic NADH/NAD+ ratio owing to limited reoxidation of reducing equivalents. Clinically, pyruvate may have a therapeutic benefit for CTLN2 patients.
Assuntos
Amônia/metabolismo , Proteínas de Ligação ao Cálcio/genética , Citrulinemia/tratamento farmacológico , Citrulinemia/metabolismo , Transportadores de Ânions Orgânicos/genética , Ácido Pirúvico/farmacologia , Ureia/metabolismo , Aminoácidos/metabolismo , Animais , Anticoagulantes/farmacologia , Ácido Aspártico/farmacologia , Ácido Cítrico/farmacologia , Citrulinemia/genética , Hiperamonemia/tratamento farmacológico , Hiperamonemia/genética , Hiperamonemia/metabolismo , Técnicas In Vitro , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Oxirredução , PerfusãoAssuntos
Proteínas de Ligação ao Cálcio/genética , Citrulinemia/genética , Expressão Gênica/fisiologia , Transportadores de Ânions Orgânicos/genética , Adulto , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/deficiência , Citrulinemia/complicações , Citrulinemia/metabolismo , Predisposição Genética para Doença , Humanos , Mutação , Transportadores de Ânions Orgânicos/deficiênciaRESUMO
OBJECTIVE: We report a risk of worsening of encephalopathy by glycerol infusion when this osmotic agent is used for the treatment of brain edema in patients with adult-onset type II citrullinemia (CTLN2) caused by citrin deficiency. PATIENTS AND METHODS: We performed a retrospective investigation of 3 patients with CTLN2 treated for brain edema at our institute: a 31-year-old male patient and a 40-year-old female patient received treatment for encephalopathy-related brain edema with 10% glycerol infusion and 20% D-mannitol, and a 40-year-old male patient received only 20% D-mannitol infusion. In addition, we also performed a retrospective study in 11 CTLN2 patients reported previously (8 patients treated with 10% glycerol, 2 treated with 10% glycerol and 20% mannitol, and 1 treated with 20% mannitol). RESULTS: The 12 patients treated with 10% glycerol, including 2 of our patients, died due to rapidly deteriorating encephalopathy and brain edema. On the other hand, the 2 patients who received only 20% D-mannitol, including one of our patients, recovered with the disappearance of brain edema. CONCLUSION: In CTLN2 patients, glycerol infusion seems to be associated with exacerbation of encephalopathy itself and only mannitol should be used for the treatment of brain edema in patients with this disorder.
Assuntos
Edema Encefálico/induzido quimicamente , Proteínas de Ligação ao Cálcio/deficiência , Citrulinemia/tratamento farmacológico , Glicerol/efeitos adversos , Transportadores de Ânions Orgânicos/deficiência , Adulto , Idade de Início , Edema Encefálico/diagnóstico por imagem , Edema Encefálico/tratamento farmacológico , Proteínas de Ligação ao Cálcio/genética , Citrulinemia/genética , Citrulinemia/metabolismo , DNA/análise , Progressão da Doença , Diuréticos Osmóticos/administração & dosagem , Diuréticos Osmóticos/uso terapêutico , Evolução Fatal , Feminino , Seguimentos , Glicerol/administração & dosagem , Humanos , Infusões Intravenosas , Masculino , Manitol/administração & dosagem , Manitol/uso terapêutico , Proteínas de Membrana Transportadoras/genética , Proteínas de Transporte da Membrana Mitocondrial , Proteínas Mitocondriais/genética , Transportadores de Ânions Orgânicos/genética , Estudos Retrospectivos , Tomografia Computadorizada por Raios XRESUMO
Citrin is a mitochondrial aspartate glutamate carrier primarily expressed in the liver, heart, and kidney. We found that adult-onset type II citrullinemia is caused by mutations in the SLC25A13 gene that encodes for citrin. In this report, we describe the frequency of SLC25A13 mutations, the roles of citrin as a member of the urea cycle and as a member of the malate-aspartate shuttle, the relationship between its functions and symptoms of citrin deficiency, and therapeutic issues.