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1.
Cell ; 184(26): 6226-6228, 2021 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-34942098

RESUMEN

Altered metabolism of tumors offers an opportunity to use metabolic interventions as a therapeutic strategy. Lien et al. demonstrate that understanding how specific diets with different carbohydrate and fat composition affect tumor metabolism is essential in order to use this opportunity efficiently.


Asunto(s)
Carbohidratos de la Dieta , Neoplasias , Dieta , Humanos , Comidas , Neoplasias/tratamiento farmacológico
2.
Nature ; 566(7744): 403-406, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30728499

RESUMEN

Most tumours have an aberrantly activated lipid metabolism1,2 that enables them to synthesize, elongate and desaturate fatty acids to support proliferation. However, only particular subsets of cancer cells are sensitive to approaches that target fatty acid metabolism and, in particular, fatty acid desaturation3. This suggests that many cancer cells contain an unexplored plasticity in their fatty acid metabolism. Here we show that some cancer cells can exploit an alternative fatty acid desaturation pathway. We identify various cancer cell lines, mouse hepatocellular carcinomas, and primary human liver and lung carcinomas that desaturate palmitate to the unusual fatty acid sapienate to support membrane biosynthesis during proliferation. Accordingly, we found that sapienate biosynthesis enables cancer cells to bypass the known fatty acid desaturation pathway that is dependent on stearoyl-CoA desaturase. Thus, only by targeting both desaturation pathways is the in vitro and in vivo proliferation of cancer cells that synthesize sapienate impaired. Our discovery explains metabolic plasticity in fatty acid desaturation and constitutes an unexplored metabolic rewiring in cancers.


Asunto(s)
Ácidos Grasos/química , Ácidos Grasos/metabolismo , Redes y Vías Metabólicas , Neoplasias/metabolismo , Neoplasias/patología , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Proliferación Celular , Ácido Graso Desaturasas/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ácidos Oléicos/metabolismo , Palmitatos/metabolismo , Ácidos Palmíticos/metabolismo , Estearoil-CoA Desaturasa/metabolismo
3.
Mol Ther ; 31(8): 2507-2523, 2023 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-37143324

RESUMEN

Age-related and chemotherapy-induced bone loss depends on cellular senescence and the cell secretory phenotype. However, the factors secreted in the senescent microenvironment that contribute to bone loss remain elusive. Here, we report a central role for the inflammatory alternative complement system in skeletal bone loss. Through transcriptomic analysis of bone samples, we identified complement factor D, a rate-limiting factor of the alternative pathway of complement, which is among the most responsive factors to chemotherapy or estrogen deficiency. We show that osteoblasts and osteocytes are major inducers of complement activation, while monocytes and osteoclasts are their primary targets. Genetic deletion of C5ar1, the receptor of the anaphylatoxin C5a, or treatment with a C5AR1 inhibitor reduced monocyte chemotaxis and osteoclast differentiation. Moreover, genetic deficiency or inhibition of C5AR1 partially prevented bone loss and osteoclastogenesis upon chemotherapy or ovariectomy. Altogether, these lines of evidence support the idea that inhibition of alternative complement pathways may have some therapeutic benefit in osteopenic disorders.


Asunto(s)
Osteoclastos , Osteogénesis , Femenino , Animales , Osteoclastos/metabolismo , Osteogénesis/genética , Osteoblastos/metabolismo , Monocitos/metabolismo , Complemento C5a/genética , Complemento C5a/metabolismo
4.
Br J Cancer ; 121(1): 51-64, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31114017

RESUMEN

BACKGROUND: Previous studies suggested that the metabolism is differently reprogrammed in the major subtypes of non-small cell lung cancer (NSCLC), squamous cell carcinomas (SCC) and adenocarcinomas (AdC). However, a comprehensive analysis of this differential metabolic reprogramming is lacking. METHODS: Publicly available gene expression data from human lung cancer samples and cell lines were analysed. Stable isotope resolved metabolomics were performed on SCC and ADC tumours in human patients and in freshly resected tumour slices. RESULTS: Analysis of multiple transcriptomics data from human samples identified a SCC-distinguishing enzyme gene signature. SCC tumours from patients infused with [U-13C]-glucose and SCC tissue slices incubated with stable isotope tracers demonstrated differential glucose and glutamine catabolism compared to AdCs or non-cancerous lung, confirming increased activity through pathways defined by the SCC metabolic gene signature. Furthermore, the upregulation of Notch target genes was a distinguishing feature of SCCs, which correlated with the metabolic signature. Notch and MYC-driven murine lung tumours recapitulated the SCC-distinguishing metabolic reprogramming. However, the differences between SCCs and AdCs disappear in established cell lines in 2D culture. CONCLUSIONS: Our data emphasise the importance of studying lung cancer metabolism in vivo. They also highlight potential targets for therapeutic intervention in SCC patients including differentially expressed enzymes that catalyse reactions in glycolysis, glutamine catabolism, serine, nucleotide and glutathione biosynthesis.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores Notch/fisiología , Adenocarcinoma del Pulmón/metabolismo , Animales , Carcinoma de Células Escamosas/metabolismo , Humanos , Ratones , Proteínas Proto-Oncogénicas c-myc/fisiología , Transcriptoma , Microambiente Tumoral
5.
FASEB J ; 29(4): 1414-25, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25550462

RESUMEN

The skeleton acts as an endocrine organ that regulates energy metabolism and calcium and phosphorous homeostasis through the secretion of osteocalcin (Oc) and fibroblast growth factor 23 (FGF23). However, evidence suggests that osteoblasts secrete additional unknown factors that contribute to the endocrine function of bone. To search for these additional factors, we generated mice with a conditional osteoblast-specific deletion of p38α MAPK known to display profound defects in bone homeostasis. Herein, we show that impaired osteoblast function is associated with a strong decrease in body weight and adiposity (P < 0.01). The differences in adiposity were not associated with diminished caloric intake, but rather reflected 20% increased energy expenditure and the up-regulation of uncoupling protein-1 (Ucp1) in white adipose tissue (WAT) and brown adipose tissue (BAT) (P < 0.05). These alterations in lipid metabolism and energy expenditure were correlated with a decrease in the blood levels of neuropeptide Y (NPY) (40% lower) rather than changes in the serum levels of insulin, Oc, or FGF23. Among all Npy-expressing tissues, only bone and primary osteoblasts showed a decline in Npy expression (P < 0.01). Moreover, the intraperitoneal administration of recombinant NPY partially restored the WAT weight and adipocyte size of p38α-deficient mice (P < 0.05). Altogether, these results further suggest that, in addition to Oc, other bone-derived signals affect WAT and energy expenditure contributing to the regulation of energy metabolism.


Asunto(s)
Tejido Adiposo/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Osteoblastos/enzimología , Adipocitos/citología , Adipocitos/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Adiposidad , Animales , Peso Corporal , Desarrollo Óseo , Tamaño de la Célula , Metabolismo Energético , Femenino , Factor-23 de Crecimiento de Fibroblastos , Regulación de la Expresión Génica , Homeostasis , Canales Iónicos/genética , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , Proteína Quinasa 14 Activada por Mitógenos/deficiencia , Proteína Quinasa 14 Activada por Mitógenos/genética , Neuropéptido Y/sangre , Neuropéptido Y/genética , Obesidad/enzimología , Obesidad/prevención & control , Osteocalcina/metabolismo , Embarazo , Transducción de Señal , Proteína Desacopladora 1 , Regulación hacia Arriba
6.
J Biol Chem ; 289(32): 22090-102, 2014 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-24973213

RESUMEN

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), encoded by the nuclear PCK2 gene, links TCA cycle intermediates and glycolytic pools through the conversion of mitochondrial oxaloacetate into phosphoenolpyruvate. In the liver PEPCK-M adjoins its profusely studied cytosolic isoform (PEPCK-C) potentiating gluconeogenesis and TCA flux. However, PEPCK-M is present in a variety of non-gluconeogenic tissues, including tumors of several origins. Despite its potential relevance to cancer metabolism, the mechanisms responsible for PCK2 gene regulation have not been elucidated. The present study demonstrates PEPCK-M overexpression in tumorigenic cells as well as the mechanism for the modulation of PCK2 abundance under several stress conditions. Amino acid limitation and ER stress inducers, conditions that activate the amino acid response (AAR) and the unfolded protein response (UPR), stimulate PCK2 gene transcription. Both the AAR and UPR lead to increased synthesis of ATF4, which mediates PCK2 transcriptional up-regulation through its binding to a putative ATF/CRE composite site within the PCK2 promoter functioning as an amino acid response element. In addition, activation of the GCN2-eIF2α-ATF4 and PERK-eIF2α-ATF4 signaling pathways are responsible for increased PEPCK-M levels. Finally, PEPCK-M knockdown using either siRNA or shRNA were sufficient to reduce MCF7 mammary carcinoma cell growth and increase cell death under glutamine deprivation or ER stress conditions. Our data demonstrate that this enzyme has a critical role in the survival program initiated upon stress and shed light on an unexpected and important role of mitochondrial PEPCK in cancer metabolism.


Asunto(s)
Neoplasias/genética , Neoplasias/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Factor de Transcripción Activador 4/metabolismo , Aminoácidos/metabolismo , Animales , Estrés del Retículo Endoplásmico , Femenino , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Ratones , Mitocondrias/enzimología , Modelos Biológicos , Células 3T3 NIH , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Respuesta de Proteína Desplegada , eIF-2 Quinasa/metabolismo
7.
Mol Cell Proteomics ; 12(8): 2111-25, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23625662

RESUMEN

Bone metastasis is the most common distant relapse in breast cancer. The identification of key proteins involved in the osteotropic phenotype would represent a major step toward the development of new prognostic markers and therapeutic improvements. The aim of this study was to characterize functional phenotypes that favor bone metastasis in human breast cancer. We used the human breast cancer cell line MDA-MB-231 and its osteotropic BO2 subclone to identify crucial proteins in bone metastatic growth. We identified 31 proteins, 15 underexpressed and 16 overexpressed, in BO2 cells compared with parental cells. We employed a network-modeling approach in which these 31 candidate proteins were prioritized with respect to their potential in metastasis formation, based on the topology of the protein-protein interaction network and differential expression. The protein-protein interaction network provided a framework to study the functional relationships between biological molecules by attributing functions to genes whose functions had not been characterized. The combination of expression profiles and protein interactions revealed an endoplasmic reticulum-thiol oxidoreductase, ERp57, functioning as a hub that retained four down-regulated nodes involved in antigen presentation associated with the human major histocompatibility complex class I molecules, including HLA-A, HLA-B, HLA-E, and HLA-F. Further analysis of the interaction network revealed an inverse correlation between ERp57 and vimentin, which influences cytoskeleton reorganization. Moreover, knockdown of ERp57 in BO2 cells confirmed its bone organ-specific prometastatic role. Altogether, ERp57 appears as a multifunctional chaperone that can regulate diverse biological processes to maintain the homeostasis of breast cancer cells and promote the development of bone metastasis.


Asunto(s)
Neoplasias Óseas/metabolismo , Neoplasias de la Mama/metabolismo , Metástasis de la Neoplasia , Proteína Disulfuro Isomerasas/metabolismo , Animales , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Ratones , Ratones SCID , Mapeo de Interacción de Proteínas , Proteoma , Transcriptoma , Vimentina/metabolismo
8.
J Biol Chem ; 288(15): 10640-51, 2013 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-23457334

RESUMEN

Reciprocal regulation of metabolism and signaling allows cells to modulate their activity in accordance with their metabolic resources. Thus, amino acids could activate signal transduction pathways that control cell metabolism. To test this hypothesis, we analyzed the effect of amino acids on fructose-2,6-bisphosphate (Fru-2,6-P2) metabolism. We demonstrate that amino acids increase Fru-2,6-P2 concentration in HeLa and in MCF7 human cells. In conjunction with this, 6-phosphofructo-2-kinase activity, glucose uptake, and lactate concentration were increased. These data correlate with the specific phosphorylation of heart 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB2) isoenzyme at Ser-483. This activation was mediated by the PI3K and p38 signaling pathways. Furthermore, Akt inactivation blocked PFKFB2 phosphorylation and Fru-2,6-P2 production, thereby suggesting that the above signaling pathways converge at Akt kinase. In accordance with these results, kinase assays showed that amino acid-activated Akt phosphorylated PFKFB2 at Ser-483 and that knockdown experiments confirmed that the increase in Fru-2,6-P2 concentration induced by amino acids was due to PFKFB2. In addition, similar effects on Fru-2,6-P2 metabolism were observed in freshly isolated rat cardiomyocytes treated with amino acids, which indicates that these effects are not restricted to human cancer cells. In these cardiomyocytes, the glucose consumption and the production of lactate and ATP suggest an increase of glycolytic flux. Taken together, these results demonstrate that amino acids stimulate Fru-2,6-P2 synthesis by Akt-dependent PFKFB2 phosphorylation and activation and show how signaling and metabolism are inextricably linked.


Asunto(s)
Aminoácidos/metabolismo , Glucólisis/fisiología , Miocitos Cardíacos/enzimología , Fosfofructoquinasa-2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Aminoácidos/genética , Animales , Activación Enzimática/fisiología , Fructosadifosfatos/genética , Fructosadifosfatos/metabolismo , Glucosa/genética , Glucosa/metabolismo , Células HEK293 , Células HeLa , Humanos , Ácido Láctico/metabolismo , Masculino , Miocitos Cardíacos/citología , Fosfofructoquinasa-2/genética , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Ratas , Ratas Sprague-Dawley , Serina/genética , Serina/metabolismo
9.
J Hepatol ; 59(1): 105-13, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23466304

RESUMEN

BACKGROUND & AIMS: Hepatic gluconeogenesis helps maintain systemic energy homeostasis by compensating for discontinuities in nutrient supply. Liver-specific deletion of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) abolishes gluconeogenesis from mitochondrial substrates, deregulates lipid metabolism and affects TCA cycle. While the mouse liver almost exclusively expresses PEPCK-C, humans equally present a mitochondrial isozyme (PEPCK-M). Despite clear relevance to human physiology, the role of PEPCK-M and its gluconeogenic potential remain unknown. Here, we test the significance of PEPCK-M in gluconeogenesis and TCA cycle function in liver-specific PEPCK-C knockout and WT mice. METHODS: The effects of the overexpression of PEPCK-M were examined by a combination of tracer studies and molecular biology techniques. Partial PEPCK-C re-expression was used as a positive control. Metabolic fluxes were evaluated in isolated livers by NMR using (2)H and (13)C tracers. Gluconeogenic potential, together with metabolic profiling, was investigated in vivo and in primary hepatocytes. RESULTS: PEPCK-M expression partially rescued defects in lipid metabolism, gluconeogenesis and TCA cycle function impaired by PEPCK-C deletion, while ∼10% re-expression of PEPCK-C normalized most parameters. When PEPCK-M was expressed in the presence of PEPCK-C, the mitochondrial isozyme amplified total gluconeogenic capacity, suggesting autonomous regulation of oxaloacetate to phosphoenolpyruvate fluxes by the individual isoforms. CONCLUSIONS: We conclude that PEPCK-M has gluconeogenic potential per se, and cooperates with PEPCK-C to adjust gluconeogenic/TCA flux to changes in substrate or energy availability, hinting at a role in the regulation of glucose and lipid metabolism in the human liver.


Asunto(s)
Gluconeogénesis/fisiología , Hígado/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Animales , Ciclo del Ácido Cítrico , Citosol/metabolismo , Expresión Génica , Gluconeogénesis/genética , Glucosa/metabolismo , Hepatocitos/metabolismo , Humanos , Metabolismo de los Lípidos , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/deficiencia , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo
10.
Nat Metab ; 5(11): 1870-1886, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37946084

RESUMEN

Tumors are intrinsically heterogeneous and it is well established that this directs their evolution, hinders their classification and frustrates therapy1-3. Consequently, spatially resolved omics-level analyses are gaining traction4-9. Despite considerable therapeutic interest, tumor metabolism has been lagging behind this development and there is a paucity of data regarding its spatial organization. To address this shortcoming, we set out to study the local metabolic effects of the oncogene c-MYC, a pleiotropic transcription factor that accumulates with tumor progression and influences metabolism10,11. Through correlative mass spectrometry imaging, we show that pantothenic acid (vitamin B5) associates with MYC-high areas within both human and murine mammary tumors, where its conversion to coenzyme A fuels Krebs cycle activity. Mechanistically, we show that this is accomplished by MYC-mediated upregulation of its multivitamin transporter SLC5A6. Notably, we show that SLC5A6 over-expression alone can induce increased cell growth and a shift toward biosynthesis, whereas conversely, dietary restriction of pantothenic acid leads to a reversal of many MYC-mediated metabolic changes and results in hampered tumor growth. Our work thus establishes the availability of vitamins and cofactors as a potential bottleneck in tumor progression, which can be exploited therapeutically. Overall, we show that a spatial understanding of local metabolism facilitates the identification of clinically relevant, tractable metabolic targets.


Asunto(s)
Neoplasias de la Mama , Humanos , Ratones , Animales , Femenino , Neoplasias de la Mama/metabolismo , Ácido Pantoténico , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/metabolismo , Vitaminas
11.
J Lipid Res ; 53(6): 1080-92, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22493093

RESUMEN

The manner in which insulin resistance impinges on hepatic mitochondrial function is complex. Although liver insulin resistance is associated with respiratory dysfunction, the effect on fat oxidation remains controversial, and biosynthetic pathways that traverse mitochondria are actually increased. The tricarboxylic acid (TCA) cycle is the site of terminal fat oxidation, chief source of electrons for respiration, and a metabolic progenitor of gluconeogenesis. Therefore, we tested whether insulin resistance promotes hepatic TCA cycle flux in mice progressing to insulin resistance and fatty liver on a high-fat diet (HFD) for 32 weeks using standard biomolecular and in vivo (2)H/(13)C tracer methods. Relative mitochondrial content increased, but respiratory efficiency declined by 32 weeks of HFD. Fasting ketogenesis became unresponsive to feeding or insulin clamp, indicating blunted but constitutively active mitochondrial ß-oxidation. Impaired insulin signaling was marked by elevated in vivo gluconeogenesis and anaplerotic and oxidative TCA cycle flux. The induction of TCA cycle function corresponded to the development of mitochondrial respiratory dysfunction, hepatic oxidative stress, and inflammation. Thus, the hepatic TCA cycle appears to enable mitochondrial dysfunction during insulin resistance by increasing electron deposition into an inefficient respiratory chain prone to reactive oxygen species production and by providing mitochondria-derived substrate for elevated gluconeogenesis.


Asunto(s)
Ciclo del Ácido Cítrico , Dieta Alta en Grasa/efectos adversos , Hígado Graso/metabolismo , Hígado Graso/patología , Resistencia a la Insulina , Animales , Respiración de la Célula , Hígado Graso/complicaciones , Hígado Graso/etiología , Gluconeogénesis , Hiperglucemia/complicaciones , Cuerpos Cetónicos/biosíntesis , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/patología , Obesidad/complicaciones , Estrés Oxidativo , Factores de Tiempo
12.
Cell Death Dis ; 13(8): 730, 2022 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-36002449

RESUMEN

On glucose restriction, epithelial cells can undergo entosis, a cell-in-cell cannibalistic process, to allow considerable withstanding to this metabolic stress. Thus, we hypothesized that reduced protein glycosylation might participate in the activation of this cell survival pathway. Glucose deprivation promoted entosis in an MCF7 breast carcinoma model, as evaluated by direct inspection under the microscope, or revealed by a shift to apoptosis + necrosis in cells undergoing entosis treated with a Rho-GTPase kinase inhibitor (ROCKi). In this context, curbing protein glycosylation defects with N-acetyl-glucosamine partially rescued entosis, whereas limiting glycosylation in the presence of glucose with tunicamycin or NGI-1, but not with other unrelated ER-stress inducers such as thapsigargin or amino-acid limitation, stimulated entosis. Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is upregulated by glucose deprivation, thereby enhancing cell survival. Therefore, we presumed that PEPCK-M could play a role in this process by offsetting key metabolites into glycosyl moieties using alternative substrates. PEPCK-M inhibition using iPEPCK-2 promoted entosis in the absence of glucose, whereas its overexpression inhibited entosis. PEPCK-M inhibition had a direct role on total protein glycosylation as determined by Concanavalin A binding, and the specific ratio of fully glycosylated LAMP1 or E-cadherin. The content of metabolites, and the fluxes from 13C-glutamine label into glycolytic intermediates up to glucose-6-phosphate, and ribose- and ribulose-5-phosphate, was dependent on PEPCK-M content as measured by GC/MS. All in all, we demonstrate for the first time that protein glycosylation defects precede and initiate the entosis process and implicates PEPCK-M in this survival program to dampen the consequences of glucose deprivation. These results have broad implications to our understanding of tumor metabolism and treatment strategies.


Asunto(s)
Neoplasias de la Mama , Entosis , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Femenino , Glucosa/metabolismo , Glicosilación , Humanos
13.
Cancer Metab ; 9(1): 1, 2021 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-33413684

RESUMEN

BACKGROUND: Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is expressed in all cancer types examined and in neuroprogenitor cells. The gene is upregulated by amino acid limitation and ER-stress in an ATF4-dependent manner, and its activity modulates the PEP/Ca2+ signaling axis, providing clear arguments for a functional relationship with metabolic adaptations for cell survival. Despite its potential relevance to cancer metabolism, the mechanisms responsible for its pro-survival activity have not been completely elucidated. METHODS: [U-13C]glutamine and [U-13C]glucose labeling of glycolytic and TCA cycle intermediates and their anabolic end-products was evaluated quantitatively using LC/MS and GC/MS in conditions of abundant glucose and glucose limitation in loss-of-function (shRNA) and gain-of-function (lentiviral constitutive overexpression) HeLa cervix carcinoma cell models. Cell viability was assessed in conjunction with various glucose concentrations and in xenografts in vivo. RESULTS: PEPCK-M levels linearly correlated with [U-13C]glutamine label abundance in most glycolytic and TCA cycle intermediate pools under nutritional stress. In particular, serine, glycine, and proline metabolism, and the anabolic potential of the cell, were sensitive to PEPCK-M activity. Therefore, cell viability defects could be rescued by supplementing with an excess of those amino acids. PEPCK-M silenced or inhibited cells in the presence of abundant glucose showed limited growth secondary to TCA cycle blockade and increased ROS. In limiting glucose conditions, downregulation of PKC-ζ tumor suppressor has been shown to enhance survival. Consistently, HeLa cells also sustained a survival advantage when PKC-ζ tumor suppressor was downregulated using shRNA, but this advantage was abolished in the absence of PEPCK-M, as its inhibition restores cell growth to control levels. The relationship between these two pathways is also highlighted by the anti-correlation observed between PEPCK-M and PKC-ζ protein levels in all clones tested, suggesting co-regulation in the absence of glucose. Finally, PEPCK-M loss negatively impacted on anchorage-independent colony formation and xenograft growth in vivo. CONCLUSIONS: All in all, our data suggest that PEPCK-M might participate in the mechanisms to regulate proteostasis in the anabolic and stalling phases of tumor growth. We provide molecular clues into the clinical relevance of PEPCK-M as a mechanism of evasion of cancer cells in conditions of nutrient stress.

14.
Nat Metab ; 2(4): 335-350, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32694609

RESUMEN

Plasticity of cancer metabolism can be a major obstacle to efficient targeting of tumour-specific metabolic vulnerabilities. Here, we identify the compensatory mechanisms following the inhibition of major pathways of central carbon metabolism in c-MYC-induced liver tumours. We find that, while inhibition of both glutaminase isoforms (Gls1 and Gls2) in tumours considerably delays tumourigenesis, glutamine catabolism continues, owing to the action of amidotransferases. Synergistic inhibition of both glutaminases and compensatory amidotransferases is required to block glutamine catabolism and proliferation of mouse and human tumour cells in vitro and in vivo. Gls1 deletion is also compensated for by glycolysis. Thus, co-inhibition of Gls1 and hexokinase 2 significantly affects Krebs cycle activity and tumour formation. Finally, the inhibition of biosynthesis of either serine (Psat1-KO) or fatty acid (Fasn-KO) is compensated for by uptake of circulating nutrients, and dietary restriction of both serine and glycine or fatty acids synergistically suppresses tumourigenesis. These results highlight the high flexibility of tumour metabolism and demonstrate that either pharmacological or dietary targeting of metabolic compensatory mechanisms can improve therapeutic outcomes.


Asunto(s)
Neoplasias Hepáticas/metabolismo , Animales , Proliferación Celular , Glucosa/metabolismo , Glutaminasa/antagonistas & inhibidores , Glutaminasa/genética , Glutamina/metabolismo , Humanos , Neoplasias Hepáticas/patología , Ratones , Proteínas Proto-Oncogénicas c-myc/metabolismo
15.
J Physiol Biochem ; 73(1): 89-98, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27785616

RESUMEN

Whole body cytosolic phosphoenolpyruvate carboxykinase knockout (PEPCK-C KO) mice die early after birth with profound hypoglycemia therefore masking the role of PEPCK-C in adult, non-gluconeogenic tissues where it is expressed. To investigate whether PEPCK-C deletion in the liver was critically responsible for the hypoglycemic phenotype, we reexpress this enzyme in the liver of PEPCK-C KO pups by early postnatal administration of PEPCK-C-expressing adenovirus. This maneuver was sufficient to partially rescue hypoglycemia and allow the pups to survive and identifies the liver as a critical organ, and hypoglycemia as the critical pathomechanism, leading to early postnatal death in the whole-body PEPCK-C knockout mice. Pathology assessment of survivors also suggest a possible role for PEPCK-C in lung maturation and muscle metabolism.


Asunto(s)
Errores Innatos del Metabolismo de los Carbohidratos/veterinaria , Hipoglucemia/prevención & control , Hepatopatías/veterinaria , Hígado/enzimología , Pulmón/metabolismo , Músculo Esquelético/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/deficiencia , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/enzimología , Encéfalo/metabolismo , Encéfalo/patología , Errores Innatos del Metabolismo de los Carbohidratos/enzimología , Errores Innatos del Metabolismo de los Carbohidratos/fisiopatología , Errores Innatos del Metabolismo de los Carbohidratos/terapia , Cruzamientos Genéticos , Técnicas de Transferencia de Gen , Gluconeogénesis , Heterocigoto , Hipoglucemia/etiología , Hipoglucemia/metabolismo , Hipoglucemia/patología , Gotas Lipídicas/metabolismo , Gotas Lipídicas/patología , Metabolismo de los Lípidos , Lipidosis/etiología , Hígado/metabolismo , Hígado/patología , Hepatopatías/enzimología , Hepatopatías/fisiopatología , Hepatopatías/terapia , Pulmón/enzimología , Pulmón/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Neuronas/enzimología , Neuronas/metabolismo , Neuronas/patología , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/uso terapéutico , Proteínas Recombinantes/metabolismo
16.
Cancer Res ; 77(16): 4355-4364, 2017 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-28630053

RESUMEN

Different pyruvate kinase isoforms are expressed in a tissue-specific manner, with pyruvate kinase M2 (PKM2) suggested to be the predominant isoform in proliferating cells and cancer cells. Because of differential regulation of enzymatic activities, PKM2, but not PKM1, has been thought to favor cell proliferation. However, the role of PKM2 in tumorigenesis has been recently challenged. Here we report that increased glucose catabolism through glycolysis and increased pyruvate kinase activity in c-MYC-driven liver tumors are associated with increased expression of both PKM1 and PKM2 isoforms and decreased expression of the liver-specific isoform of pyruvate kinase, PKL. Depletion of PKM2 at the time of c-MYC overexpression in murine livers did not affect c-MYC-induced tumorigenesis and resulted in liver tumor formation with decreased pyruvate kinase activity and decreased catabolism of glucose into alanine and the Krebs cycle. An increased PKM1/PKM2 ratio by ectopic PKM1 expression further decreased glucose flux into serine biosynthesis and increased flux into lactate and the Krebs cycle, resulting in reduced total levels of serine. However, these changes also did not affect c-MYC-induced liver tumor development. These results suggest that increased expression of PKM2 is not required to support c-MYC-induced tumorigenesis in the liver and that various PKM1/PKM2 ratios and pyruvate kinase activities can sustain glucose catabolism required for this process. Cancer Res; 77(16); 4355-64. ©2017 AACR.


Asunto(s)
Glucosa/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Piruvato Quinasa/metabolismo , Animales , Genes myc , Isoenzimas , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Ratones , Fosforilación , Proteínas Proto-Oncogénicas c-myc/genética , Piruvato Quinasa/genética
17.
Int J Endocrinol ; 2011: 707928, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22194744

RESUMEN

Recent reports point out the importance of the complex GK-GKRP in controlling glucose and lipid homeostasis. Several GK mutations affect GKRP binding, resulting in permanent activation of the enzyme. We hypothesize that hepatic overexpression of a mutated form of GK, GK(A456V), described in a patient with persistent hyperinsulinemic hypoglycemia of infancy (PHHI) and could provide a model to study the consequences of GK-GKRP deregulation in vivo. GK(A456V) was overexpressed in the liver of streptozotocin diabetic mice. Metabolite profiling in serum and liver extracts, together with changes in key components of glucose and lipid homeostasis, were analyzed and compared to GK wild-type transfected livers. Cell compartmentalization of the mutant but not the wild-type GK was clearly affected in vivo, demonstrating impaired GKRP regulation. GK(A456V) overexpression markedly reduced blood glucose in the absence of dyslipidemia, in contrast to wild-type GK-overexpressing mice. Evidence in glucose utilization did not correlate with increased glycogen nor lactate levels in the liver. PEPCK mRNA was not affected, whereas the mRNA for the catalytic subunit of glucose-6-phosphatase was upregulated ~4 folds in the liver of GK(A456V)-treated animals, suggesting that glucose cycling was stimulated. Our results provide new insights into the complex GK regulatory network and validate liver-specific GK activation as a strategy for diabetes therapy.

18.
Diabetes ; 57(8): 2199-210, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18443203

RESUMEN

OBJECTIVE: Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; encoded by Pck1) catalyzes the first committed step in gluconeogenesis. Extensive evidence demonstrates a direct correlation between PEPCK-C activity and glycemia control. Therefore, we aimed to evaluate the metabolic impact and their underlying mechanisms of knocking down hepatic PEPCK-C in a type 2 diabetic model. RESEARCH DESIGN AND METHODS: PEPCK-C gene targeting was achieved using adenovirus-transduced RNAi. The study assessed several clinical symptoms of diabetes and insulin signaling in peripheral tissues, in addition to changes in gene expression, protein, and metabolites in the liver. Liver bioenergetics was also evaluated. RESULTS: Treatment resulted in reduced PEPCK-C mRNA and protein. After treatment, improved glycemia and insulinemia, lower triglyceride, and higher total and HDL cholesterol were measured. Unsterified fatty acid accumulation was observed in the liver, in the absence of de novo lipogenesis. Despite hepatic lipidosis, treatment resulted in improved insulin signaling in the liver, muscle, and adipose tissue. O(2) consumption measurements in isolated hepatocytes demonstrated unaltered mitochondrial function and a consequent increased cellular energy charge. Key regulatory factors (FOXO1, hepatocyte nuclear factor-4alpha, and peroxisome proliferator-activated receptor-gamma coactivator [PGC]-1alpha) and enzymes (G6Pase) implicated in gluconeogenesis were downregulated after treatment. Finally, the levels of Sirt1, a redox-state sensor that modulates gluconeogenesis through PGC-1alpha, were diminished. CONCLUSIONS: Our observations indicate that silencing PEPCK-C has direct impact on glycemia control and energy metabolism and provides new insights into the potential significance of the enzyme as a therapeutic target for the treatment of diabetes.


Asunto(s)
Diabetes Mellitus Tipo 2/patología , Dislipidemias/patología , Silenciador del Gen , Resistencia a la Insulina , Hígado/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Animales , Glucemia/metabolismo , Western Blotting , Citosol/enzimología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Dislipidemias/sangre , Dislipidemias/genética , Metabolismo Energético/genética , Técnica del Anticuerpo Fluorescente , Gluconeogénesis/genética , Prueba de Tolerancia a la Glucosa , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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