RESUMEN
The mechanisms of NASH development in the context of age and genetics are not fully elucidated. This study investigates the age-dependent liver defects during NASH development in mice with heterozygous deletion of Pcyt2 (Pcyt2+/-), the rate limiting enzyme in phosphatidylethanolamine (PE) synthesis. Further, the therapeutic potential of Pcyt2 substrate, phosphoethanolamine (PEtn), is examined. Pcyt2+/- were investigated at 2 and 6-8 months (mo) of age and in addition, 6-mo old Pcyt2+/- with developed NASH were supplemented with PEtn for 8 weeks and glucose and fatty acid metabolism, insulin signaling, and inflammation were examined. Heterozygous ablation of Pcyt2 causes changes in liver metabolic regulators from young age, prior to the development of liver disease which does not occur until adulthood. Only older Pcyt2+/- experiences perturbed glucose and fatty acid metabolism. Older Pcyt2+/- liver develops NASH characterized by increased glucose production, accumulation of TAG and glycogen, and increased inflammation. Supplementation with PEtn reverses Pcyt2+/- steatosis, inflammation, and other aspects of NASH, showing that was directly caused by Pcyt2 deficiency. Pcyt2 deficiency is a novel mechanism of metabolic dysregulation due to reduced membrane ethanolamine phospholipid synthesis, and the metabolite PEtn offers therapeutic potential for NASH reversion.
Asunto(s)
Etanolaminas/administración & dosificación , Resistencia a la Insulina , Enfermedad del Hígado Graso no Alcohólico/enzimología , ARN Nucleotidiltransferasas/deficiencia , Envejecimiento , Animales , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , ARN Nucleotidiltransferasas/genéticaRESUMEN
BACKGROUND & AIMS: Since human induced pluripotent stem cells (iPSCs) develop into hepatic organoids through stages that resemble human embryonic liver development, they can be used to study developmental processes and disease pathology. Therefore, we examined the early stages of hepatic organoid formation to identify key pathways affecting early liver development. METHODS: Single-cell RNA-sequencing and metabolomic analysis was performed on developing organoid cultures at the iPSC, hepatoblast (day 9) and mature organoid stage. The importance of the phosphatidylethanolamine biosynthesis pathway to early liver development was examined in developing organoid cultures using iPSC with a CRISPR-mediated gene knockout and an over the counter medication (meclizine) that inhibits the rate-limiting enzyme in this pathway. Meclizine's effect on the growth of a human hepatocarcinoma cell line in a xenotransplantation model and on the growth of acute myeloid leukemia cells in vitro was also examined. RESULTS: Transcriptomic and metabolomic analysis of organoid development indicated that the phosphatidylethanolamine biosynthesis pathway is essential for early liver development. Unexpectedly, early hepatoblasts were selectively sensitive to the cytotoxic effect of meclizine. We demonstrate that meclizine could be repurposed for use in a new synergistic combination therapy for primary liver cancer: a glycolysis inhibitor reprograms cancer cell metabolism to make it susceptible to the cytotoxic effect of meclizine. This combination inhibited the growth of a human liver carcinoma cell line in vitro and in a xenotransplantation model, without causing significant side effects. This drug combination was also highly active against acute myeloid leukemia cells. CONCLUSION: Our data indicate that phosphatidylethanolamine biosynthesis is a targetable pathway for cancer; meclizine may have clinical efficacy as a repurposed anti-cancer drug when used as part of a new combination therapy. LAY SUMMARY: The early stages of human liver development were modeled using human hepatic organoids. We identified a pathway that was essential for early liver development. Based upon this finding, a novel combination drug therapy was identified that could be used to treat primary liver cancer and possibly other types of cancer.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Leucemia Mieloide Aguda/sangre , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Meclizina/administración & dosificación , Fosfatidiletanolaminas/antagonistas & inhibidores , Fosfatidiletanolaminas/biosíntesis , Piridinas/administración & dosificación , Quinolinas/administración & dosificación , Adulto , Anciano , Animales , Carcinoma Hepatocelular/patología , Supervivencia Celular/efectos de los fármacos , Quimioterapia Combinada/métodos , Femenino , Técnicas de Inactivación de Genes , Glucólisis/efectos de los fármacos , Células Hep G2 , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Hígado/embriología , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Persona de Mediana Edad , Organogénesis/efectos de los fármacos , Organogénesis/genética , Organoides/efectos de los fármacos , Organoides/metabolismo , ARN Nucleotidiltransferasas/deficiencia , ARN Nucleotidiltransferasas/genética , Estudios Retrospectivos , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Fosfatidiletanolaminas/biosíntesis , ARN Nucleotidiltransferasas/genética , Paraplejía Espástica Hereditaria/genética , Adolescente , Alelos , Animales , Atrofia , Encéfalo/patología , Niño , Preescolar , Discapacidades del Desarrollo/genética , Epilepsia/genética , Femenino , Técnicas de Inactivación de Genes , Variación Genética , Humanos , Lipidómica , Masculino , Ratones , ARN Nucleotidiltransferasas/deficiencia , Adulto Joven , Pez CebraRESUMEN
In Bacillus subtilis, the tRNACys lacks an encoded CCA 3' end. To gain insight into the role of CCAase and RNases in tRNACys processing, several mutant strains were generated. Northern blot and RT-PCR results suggest that enzymes other than CCAase can participate in CCA addition at the 3' end of the immature tRNACys.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , ARN Nucleotidiltransferasas/deficiencia , ARN Bacteriano/genética , ARN de Transferencia de Cisteína/genética , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Conformación de Ácido Nucleico , ARN Nucleotidiltransferasas/genética , Procesamiento Postranscripcional del ARN , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN de Transferencia de Cisteína/química , ARN de Transferencia de Cisteína/metabolismoRESUMEN
Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.
Asunto(s)
Encefalopatías Metabólicas Innatas/genética , Tronco Encefálico/metabolismo , Tronco Encefálico/virología , ARN/química , ARN/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Encefalopatías Metabólicas Innatas/patología , Tronco Encefálico/patología , Encefalitis Viral/genética , Escherichia coli/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/virología , Herpesvirus Humano 1 , Humanos , Interferones/metabolismo , Intrones/genética , Masculino , Ratones , Proteínas Mutantes/metabolismo , Mutación/genética , Sistemas de Lectura Abierta/genética , Linaje , ARN Nucleotidiltransferasas/química , ARN Nucleotidiltransferasas/deficiencia , ARN Nucleotidiltransferasas/genética , Receptor Toll-Like 3/metabolismo , Replicación ViralRESUMEN
Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not required for early steps in HIV cDNA formation but is necessary for synthesis of intermediate and late cDNA products. To further characterize this effect, we evaluated the topology of the 5' end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 synthesis. Cells were transfected with DBR1 short hairpin RNA (shRNA) followed 48 h later by infection with an HIV-1-derived vector containing an RNase H-deficient reverse transcriptase (RT). RNA was isolated at several times postinfection and treated with various RNA-modifying enzymes prior to rapid amplification of 5' cDNA ends (5' RACE) for HIV-1 RNA and quantitative reverse transcriptase PCR (qRT-PCR). In infected cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5' ends at all time points. The difference in detection of free HIV-1 RNA 5' ends in infected DBR1 knockdown versus control cells was eliminated by in vitro incubation of infected cell RNAs with yeast or human DBR1 enzyme prior to 5' RACE and qRT-PCR. This was dependent on the 2'-5' phosphatase activity of DBR1, since it did not occur when we used the catalytically inactive DBR1(N85A) mutant. Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results provide evidence for formation of a lariat-like structure involving the 5' end of HIV-1 RNA during an early step in infection and the involvement of DBR1 in resolving it.IMPORTANCE Our findings support a new view of the early steps in HIV genome replication. We show that the HIV genomic RNA is rapidly decapped and forms a lariat-like structure after entering a cell. The lariat-like structure is subsequently resolved by the cellular enzyme DBR1, leaving a 5' phosphate. This pathway is similar to the formation and resolution of pre-mRNA intron lariats and therefore suggests that similar mechanisms may be used by HIV. Our work therefore opens a new area of investigation in HIV replication and may ultimately uncover new targets for inhibiting HIV replication and for preventing the development of AIDS.
Asunto(s)
Genoma Viral , VIH-1/genética , Caperuzas de ARN/química , ARN Nucleotidiltransferasas/genética , ARN Viral/química , Transcripción Reversa , Células HEK293 , VIH-1/química , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Nucleotidiltransferasas/deficiencia , ARN Nucleotidiltransferasas/metabolismo , ARN Nucleotidiltransferasas/farmacología , Precursores del ARN/química , Empalme del ARN , ARN Interferente Pequeño , ARN Viral/metabolismo , Saccharomyces cerevisiae/genética , Replicación ViralRESUMEN
It was hypothesized that choline supplementation in insulin resistant (IR) CTP:phosphoethanolamine cytidylyltransferase deficient (Pcyt2+/-) mice would ameliorate muscle function by remodeling glucose and fatty acid (FA) metabolism. Pcyt2+/- mice either received no treatment or were allowed access to 2 mg/mL choline in drinking water for 4 weeks. Skeletal muscle was harvested from choline treated and untreated mice. Lipid analysis and metabolic gene expression and signaling pathways were compared between untreated Pcyt2+/- mice, treated Pcyt2+/- mice, and Pcyt2+/+ mice. The major positive effect of choline supplementation on IR muscle was the reduction of glucose utilization for FA and triglyceride (TAG) synthesis and increased muscle glucose storage as glycogen. Choline reduced the expression of genes for FA and TAG formation (Scd1, Fas, Srebp1c, Dgat1/2), upregulated the genes for FA oxidation (Cpt1, Pparα, Pgc1α), and had minor effects on phospholipid and lipolysis genes. Pcyt2+/- muscle had reduced insulin signaling (IRS1), autophagy (LC3), and choline transport (CTL1) proteins that were restored by choline treatment. Additionally, choline activated AMPK and Akt while inhibiting mTORC1 phosphorylation. These data established that choline supplementation could restore muscle glucose metabolism by reducing lipogenesis and improving mitochondrial and intracellular signaling for protein and energy metabolism in insulin resistant Pcyt2 deficient mice.
Asunto(s)
Adaptación Fisiológica/efectos de los fármacos , Colina/farmacología , Resistencia a la Insulina , Lipogénesis/efectos de los fármacos , ARN Nucleotidiltransferasas/genética , Adaptación Fisiológica/genética , Administración Oral , Animales , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Glucosa/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Lipogénesis/genética , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Músculo Esquelético , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , ARN Nucleotidiltransferasas/deficiencia , Transducción de Señal , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
Choline plays a critical role in systemic lipid metabolism and hepatic function. Here we conducted a series of experiments to investigate the effect of choline supplementation on metabolically altered Pcyt2(+/-) mice. In Pcyt2(+/-) mice, the membrane phosphatidylethanolamine (PE) turnover is reduced and the formation of fatty acids (FA) and triglycerides (TAG) increased, resulting in hypertriglyceridemia, liver steatosis and obesity. One month of choline supplementation reduced the incorporation of FA into TAG and facilitated TAG degradation in Pcyt2(+/-) adipocytes, plasma and liver. Choline particularly stimulated adipocyte and liver TAG lipolysis by specific lipases (ATGL, LPL and HSL) and inhibited TAG formation by DGAT1 and DGAT2. Choline also activated the liver AMPK and mitochondrial FA oxidation gene PPARα and reduced the FA synthesis genes SREBP1, SCD1 and FAS. Liver (HPLC) and plasma (tandem mass spectroscopy and (1)H-NMR) metabolite profiling established that Pcyt2(+/-) mice have reduced membrane cholesterol/sphingomyelin ratio and the homocysteine/methionine cycle that were improved by choline supplementation. These data suggest that supplementary choline is beneficial for restoring FA and TAG homeostasis under conditions of obesity caused by impaired PE synthesis.
Asunto(s)
Colina/farmacología , Hígado/efectos de los fármacos , ARN Nucleotidiltransferasas/deficiencia , Proteínas Quinasas Activadas por AMP/metabolismo , Acilación , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Carnitina/análogos & derivados , Suplementos Dietéticos , Ácidos Grasos/genética , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Ratones Mutantes , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , ARN Nucleotidiltransferasas/genética , Triglicéridos/sangre , Triglicéridos/metabolismo , Aumento de Peso/efectos de los fármacosRESUMEN
Phosphatidylethanolamine is an important inner-leaflet phospholipid, and CTP:phosphoethanolamine cytidylyltransferase-Pcyt2 acts as the main regulator of the de novo phosphatidylethanolamine synthesis from ethanolamine and diacylglycerol. Complete deletion of the mouse Pcyt2 gene is embryonic lethal, and the single-allele deficiency leads to development of the metabolic syndrome phenotype, including liver steatosis, hypertriglyceridemia, obesity, and insulin resistance. This study aimed to specifically elucidate the mechanisms of hypertriglyceridemia in Pcyt2 heterozygous mice (Pcyt2(+/-)). Evidence here shows that unlike 8 week-old mice, 32 week- and 42 week-old Pcyt2(+/-) mice experience increased VLDL secretion and liver microsomal triglyceride transfer protein activity. Older Pcyt2(+/-) mice also demonstrate increased levels of postprandial plasma TAGs, increased stimulation of genes responsible for intestinal lipid absorption, transport and chylomicron secretion, and dramatically elevated plasma Angptl4, apoB-100, and apoB-48 content. In addition, plasma HL and LPL activities and TAG clearance following a lipid challenge were significantly reduced in Pcyt2(+/-) mice relative to control littermates. Collectively, these results establish that the hypertriglyceridemia that accompanies Pcyt2 deficiency is the result of multiple metabolic adaptations, including elevated hepatic and intestinal lipoprotein secretion and stimulated expression and/or activity of genes involved in lipid absorption and transport and lipoprotein assembly, together with reduced plasma TAG clearance and utilization with peripheral tissues.
Asunto(s)
Hipertrigliceridemia/enzimología , ARN Nucleotidiltransferasas/deficiencia , Proteína 4 Similar a la Angiopoyetina , Angiopoyetinas/genética , Animales , Apolipoproteínas B/sangre , Proteínas Portadoras/metabolismo , Quilomicrones/biosíntesis , Quilomicrones/metabolismo , Ácidos Grasos/metabolismo , Regulación Enzimológica de la Expresión Génica , Homeostasis , Hipertrigliceridemia/sangre , Hipertrigliceridemia/metabolismo , Absorción Intestinal/genética , Mucosa Intestinal/metabolismo , Lipasa/sangre , Lipoproteína Lipasa/sangre , Lipoproteína Lipasa/genética , Hígado/metabolismo , Ratones , Periodo Posprandial , Triglicéridos/sangre , Triglicéridos/metabolismoRESUMEN
Phosphoethanolamine cytidylyltransferase (ECT) catalyzes the rate-controlling step in a major pathway for the synthesis of phosphatidylethanolamine (PtdEtn). Hepatocyte-specific deletion of the ECT gene in mice resulted in normal appearing animals without overt signs of liver injury or inflammation. The molecular species of PtdEtn in the ECT-deficient livers were significantly altered compared with controls and matched the composition of the phosphatidylserine (PtdSer) pool, illustrating the complete reliance on the PtdSer decarboxylase pathway for PtdEtn synthesis. PtdSer structure was controlled by the substrate specificity of PtdSer synthase that selectively converted phosphatidylcholine molecular species containing stearate paired with a polyunsaturated fatty acid to PtdSer. There was no evidence for fatty acid remodeling of PtdEtn. The elimination of diacylglycerol utilization by the CDP-ethanolamine pathway led to a 10-fold increase in triacylglycerols in the ECT-deficient hepatocytes that became engorged with lipid droplets. Triacylglycerol accumulation was associated with a significant elevation in the expression of the transcription factors and target genes that drive de novo lipogenesis. The absence of the ECT pathway for diacylglycerol utilization at the endoplasmic reticulum triggers increased fatty acid synthesis to support the formation of triacylglycerols leading to liver steatosis.
Asunto(s)
Citidina Difosfato/metabolismo , Etanolamina/metabolismo , Homeostasis , Metabolismo de los Lípidos , Hígado/metabolismo , ARN Nucleotidiltransferasas/metabolismo , Animales , Hígado Graso/genética , Hígado Graso/metabolismo , Femenino , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Homeostasis/genética , Metabolismo de los Lípidos/genética , Lipogénesis/genética , Masculino , Ratones , Especificidad de Órganos , Fosfatidiletanolaminas/biosíntesis , ARN Nucleotidiltransferasas/deficiencia , ARN Nucleotidiltransferasas/genética , ARN Mensajero/metabolismo , Transcripción Genética , Triglicéridos/metabolismoRESUMEN
The CDP-ethanolamine pathway is responsible for the de novo biosynthesis of ethanolamine phospholipids, where CDP-ethanolamine is coupled with diacylglycerols to form phosphatidylethanolamine. We have disrupted the mouse gene encoding CTP:phosphoethanolamine cytidylyltransferase, Pcyt2, the main regulatory enzyme in this pathway. Intercrossings of Pcyt2(+/-) animals resulted in small litter sizes and unexpected Mendelian frequencies, with no null mice genotyped. The Pcyt2(-/-) embryos die after implantation, prior to embryonic day 8.5. Examination of mRNA expression, protein content, and enzyme activity in Pcyt2(+/-) animals revealed the anticipated 50% decrease due to the gene dosage effect but rather a 20 to 35% decrease. [(14)C]ethanolamine radiolabeling of hepatocytes, liver, heart, and brain corroborated Pcyt2 gene expression and activity data and showed a decreased rate of phosphatidylethanolamine biosynthesis in heterozygotes. Total phospholipid content was maintained in Pcyt2(+/-) tissues; however, this was not due to compensatory increases in the decarboxylation of phosphatidylserine. These results establish the necessity of Pcyt2 for murine development and demonstrate that a single Pcyt2 allele in heterozygotes can maintain phospholipid homeostasis.