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1.
Metabolism ; 114: 154419, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33161019

RESUMO

BACKGROUND: Glycolysis controls mTORC1 signaling and protein synthesis. In skeletal muscle, glucose metabolism increases with both exercise/contraction intensity and volume, and therefore, high-intensity muscle contraction (HiMC) such as resistance exercise facilitates glycolysis including glucose uptake and glycogen breakdown. However, it is unknown whether glycolysis regulates HiMC-induced mTORC1 activation and increase in protein synthesis. METHODS: To determine whether glycolysis regulates basal and HiMC-induced mTORC1 signaling and protein synthesis, we employed 2-deoxyglucose (2-DG) to inhibit glycolysis and isometrically contracted the gastrocnemius muscle of Sprague Dawley rats using percutaneous electrical stimulation. RESULTS: Inhibition of glycolysis by 2-DG inhibited basal phosphorylation of p70S6K and 4E-BP1 (downstream targets of mTORC1) and protein synthesis (all P < 0.05) independent of AMPK phosphorylation. AMPK phosphorylation was comparably increased after HiMC at 0 h post HiMC and returned to basal levels 6 h post HiMC in both vehicle- and 2-DG-treated groups. Glycolysis inhibition attenuated muscle contraction-induced phosphorylation of 4E-BP1 at 6 h post HiMC (P < 0.05) but not p70S6K phosphorylation and protein synthesis. CONCLUSION: Although glycolysis is involved in basal but not HiMC-induced muscle protein synthesis, it regulates both basal and HiMC-induced mTORC1 signaling, and may play key roles in skeletal muscle adaptation to HiMC.


Assuntos
Desoxiglucose/farmacologia , Glicólise/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Adenilato Quinase/metabolismo , Animais , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo
2.
Metabolism ; 116: 154463, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33309713

RESUMO

OBJECTIVES: GDI1 gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human GDI1 are responsible for intellectual disability (ID). In mice with ablated Gdi1, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens if the deletion of αGDI expression is restricted to neurons. However, whether astrocytes, key homeostasis providing neuroglial cells, supporting neurons via aerobic glycolysis, contribute to this cognitive impairment is unclear. METHODS: We carried out proteomic analysis and monitored [18F]-fluoro-2-deoxy-d-glucose uptake into brain slices of Gdi1 knockout and wild type control mice. d-Glucose utilization at single astrocyte level was measured by the Förster Resonance Energy Transfer (FRET)-based measurements of cytosolic cyclic AMP, d-glucose and L-lactate, evoked by agonists selective for noradrenaline and L-lactate receptors. To test the role of astrocyte-resident processes in disease phenotype, we generated an inducible Gdi1 knockout mouse carrying the Gdi1 deletion only in adult astrocytes and conducted behavioural tests. RESULTS: Proteomic analysis revealed significant changes in astrocyte-resident glycolytic enzymes. Imaging [18F]-fluoro-2-deoxy-d-glucose revealed an increased d-glucose uptake in Gdi1 knockout tissue versus wild type control mice, consistent with the facilitated d-glucose uptake determined by FRET measurements. In mice with Gdi1 deletion restricted to astrocytes, a selective and significant impairment in working memory was recorded, which was rescued by inhibiting glycolysis by 2-deoxy-d-glucose injection. CONCLUSIONS: These results reveal a new astrocyte-based mechanism in neurodevelopmental disorders and open a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice.


Assuntos
Desoxiglucose/farmacologia , Glicólise/efeitos dos fármacos , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Deficiência Intelectual/genética , Transtornos da Memória/prevenção & controle , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Desoxiglucose/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Glucose/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/deficiência , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Transtornos da Memória/genética , Camundongos , Camundongos Knockout
3.
Int J Radiat Biol ; 96(11): 1323-1328, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32910699

RESUMO

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pandemic disease and is the major cause of deaths worldwide. The clinical complexities (inflammation, cytokine storm, and multi-organ dysfunction) associated with COVID-19 poses constraints to effective management of critically ill COVID-19 patients. Low dose radiation therapy (LDRT) has been evaluated as a potential therapeutic modality for COVID-19 pneumonia. However, due to heterogeneity in disease manifestation and inter-individual variations, effective planning for LDRT is limited for this large-scale event. 2-deoxy-D-glucose (2-DG) has emerged as a polypharmacological agent for COVID-19 treatment due to its effects on the glycolytic pathway, anti-inflammatory action, and interaction with viral proteins. We suggest that 2-DG will be a potential adjuvant to enhance the efficacy of LDRT in the treatment of COVID-19 pneumonia. Withal, azido analog of 2-DG, 2-azido-2-DG can produce rapid catastrophic oxidative stress and quell the cytokine storm in critically ill COVID-19 patients.


Assuntos
Betacoronavirus , Infecções por Coronavirus/terapia , Síndrome da Liberação de Citocina/terapia , Desoxiglucose/uso terapêutico , Pneumonia Viral/terapia , Terapia Combinada , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/metabolismo , Desoxiglucose/farmacologia , Humanos , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/metabolismo , Dosagem Radioterapêutica
4.
PLoS Genet ; 16(7): e1008484, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32673313

RESUMO

Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. Missense alleles of the HXK2, REG1, GLC7 and SNF1 genes were shown to confer significant resistance to 2-deoxyglucose and all had the potential to alter the activity and or target selection of the Snf1 kinase signaling pathway. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Addition of 2DG promotes endocytosis of the glucose transporter Hxt3. All but one of the 2DG-resistant strains reduced the 2DG-mediated hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-deoxyglucose but induction of these genes is not associated with 2DG-resistance. RNAseq analysis of the transcriptional response to 2DG showed large scale, genome-wide changes in mRNA abundance that were greatly reduced in the 2DG resistant strains. These findings suggest the common adaptive response to 2DG is to limit the magnitude of the response. Genetic studies of 2DG resistance using the dominant SNF1-G53R allele in cells that are genetically compromised in both the endocytosis and DOG pathways suggest that at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.


Assuntos
Metabolismo Energético/genética , Glucose/metabolismo , Hexoquinase/genética , Monoéster Fosfórico Hidrolases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Desoxiglucose/efeitos adversos , Desoxiglucose/farmacologia , Endocitose/efeitos dos fármacos , Endocitose/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/genética , Humanos , Mutação/genética , Proteína Fosfatase 1/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Sequenciamento Completo do Genoma
5.
PLoS Pathog ; 16(6): e1008568, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32516328

RESUMO

Human herpesvirus 6 (HHV-6) is an important immunosuppressive and immunomodulatory virus worldwide. However, whether and how HHV-6 infection influences the metabolic machinery of the host cell to provide the energy and biosynthetic resources for virus propagation remains unknown. In this study, we identified that HHV-6A infection promotes glucose metabolism in infected T cells, resulting in elevated glycolytic activity with an increase of glucose uptake, glucose consumption and lactate secretion. Furthermore, we explored the mechanisms involved in HHV-6A-mediated glycolytic activation in the infected T cells. We found increased expressions of the key glucose transporters and glycolytic enzymes in HHV-6A-infected T cells. In addition, HHV-6A infection dramatically activated AKT-mTORC1 signaling in the infected T cells and pharmacological inhibition of mTORC1 blocked HHV-6A-mediated glycolytic activation. We also found that direct inhibition of glycolysis by 2-Deoxy-D-glucose (2-DG) or inhibition of mTORC1 activity in HHV-6A-infected T cells effectively reduced HHV-6 DNA replication, protein synthesis and virion production. These results not only reveal the mechanism of how HHV-6 infection affects host cell metabolism, but also suggest that targeting the metabolic pathway could be a new avenue for HHV-6 therapy.


Assuntos
Glicólise , Herpesvirus Humano 6/metabolismo , Infecções por Roseolovirus/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , DNA Viral/biossíntese , Desoxiglucose/farmacologia , Glucose/metabolismo , Humanos , Ácido Láctico/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Infecções por Roseolovirus/tratamento farmacológico , Infecções por Roseolovirus/patologia , Linfócitos T/patologia , Linfócitos T/virologia , Proteínas Virais/biossíntese , Vírion/metabolismo
6.
Epilepsia ; 61(7): 1528-1537, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32558935

RESUMO

OBJECTIVE: Neonatal status epilepticus (SE) is a life-threatening medical emergency. Unfortunately, up to 50% of neonates with SE are resistant to current antiseizure drugs, highlighting the need for better treatments. This study aims to explore a novel metabolic approach as a potential alternative treatment to control neonatal SE, using the glycolytic inhibitor 2-deoxyglucose (2-DG). METHODS: SE was induced by pilocarpine (300 mg/kg, intraperitoneally [ip]) in neonatal Sprague Dawley rats (postnatal day 10 [P10]-P17) and was monitored by video-electroencephalography (V-EEG). After 30 minutes of SE, 2-DG or one of two conventional antiseizure drugs with different mechanisms of action, phenobarbital or levetiracetam, was administrated ip, and V-EEG recording was continued for ~60 additional minutes. The time to seizure cessation after drug injection, EEG scores, and power spectra before and after drug or saline treatment were used to assess drug effects. RESULTS: Once SE became sustained, administration of 2-DG (50, 100, or 500 mg/kg, ip) consistently stopped behavioral and electrographic seizures within 10-15 minutes; lower doses took longer (25-30 minutes) to stop SE, demonstrating a dose-dependent effect. Administration of phenobarbital (30 mg/kg, ip) or levetiracetam (100 mg/kg, ip) also stopped SE within 10-15 minutes in neonatal rats. SIGNIFICANCE: Our results suggest that the glycolysis inhibitor 2-DG acts quickly to reduce neuronal hyperexcitability and effectively suppress ongoing seizure activity, which may provide translational value in the treatment of neonatal SE.


Assuntos
Desoxiglucose/uso terapêutico , Pilocarpina/toxicidade , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/tratamento farmacológico , Animais , Animais Recém-Nascidos , Antimetabólitos/farmacologia , Antimetabólitos/uso terapêutico , Desoxiglucose/farmacologia , Eletroencefalografia/efeitos dos fármacos , Eletroencefalografia/métodos , Agonistas Muscarínicos/toxicidade , Ratos , Ratos Sprague-Dawley , Estado Epiléptico/fisiopatologia , Gravação em Vídeo/métodos
7.
Proc Natl Acad Sci U S A ; 117(18): 9932-9941, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32312819

RESUMO

Cellular starvation is typically a consequence of tissue injury that disrupts the local blood supply but can also occur where cell populations outgrow the local vasculature, as observed in solid tumors. Cells react to nutrient deprivation by adapting their metabolism, or, if starvation is prolonged, it can result in cell death. Cell starvation also triggers adaptive responses, like angiogenesis, that promote tissue reorganization and repair, but other adaptive responses and their mediators are still poorly characterized. To explore this issue, we analyzed secretomes from glucose-deprived cells, which revealed up-regulation of multiple cytokines and chemokines, including IL-6 and IL-8, in response to starvation stress. Starvation-induced cytokines were cell type-dependent, and they were also released from primary epithelial cells. Most cytokines were up-regulated in a manner dependent on NF-κB and the transcription factor of the integrated stress response ATF4, which bound directly to the IL-8 promoter. Furthermore, glutamine deprivation, as well as the antimetabolic drugs 2-deoxyglucose and metformin, also promoted the release of IL-6 and IL-8. Finally, some of the factors released from starved cells induced chemotaxis of B cells, macrophages, and neutrophils, suggesting that nutrient deprivation in the tumor environment can serve as an initiator of tumor inflammation.


Assuntos
Inflamação/genética , Interleucina-6/genética , Interleucina-8/genética , Neoplasias/metabolismo , Estresse Fisiológico/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Antimetabólitos/farmacologia , Morte Celular/efeitos dos fármacos , Desoxiglucose/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Glucose/metabolismo , Glutamina/metabolismo , Células HeLa , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Metformina/farmacologia , NF-kappa B/genética , Neoplasias/genética , Regiões Promotoras Genéticas/genética , Inanição/genética , Inanição/metabolismo , Estresse Fisiológico/imunologia
8.
Cancer Cell ; 37(4): 599-617.e7, 2020 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-32243837

RESUMO

Epigenetic modifiers frequently harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Histone methyltransferase KMT2D (a COMPASS-like enzyme, also called MLL4) is among the most highly inactivated epigenetic modifiers in lung cancer. Here, we show that lung-specific loss of Kmt2d promotes lung tumorigenesis in mice and upregulates pro-tumorigenic programs, including glycolysis. Pharmacological inhibition of glycolysis preferentially impedes tumorigenicity of human lung cancer cells bearing KMT2D-inactivating mutations. Mechanistically, Kmt2d loss widely impairs epigenomic signals for super-enhancers/enhancers, including the super-enhancer for the circadian rhythm repressor Per2. Loss of Kmt2d decreases expression of PER2, which regulates multiple glycolytic genes. These findings indicate that KMT2D is a lung tumor suppressor and that KMT2D deficiency confers a therapeutic vulnerability to glycolytic inhibitors.


Assuntos
Adenocarcinoma de Pulmão/patologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Desoxiglucose/farmacologia , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Glicólise , Histona-Lisina N-Metiltransferase/fisiologia , Proteína de Leucina Linfoide-Mieloide/fisiologia , Proteínas de Neoplasias/antagonistas & inibidores , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/metabolismo , Animais , Antimetabólitos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Camundongos Nus , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Life Sci ; 248: 117474, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32112869

RESUMO

BACKGROUND/OBJECTIVES: Nicotinamide N-methyltransferase (NNMT) is a novel regulator of energy homeostasis in adipocytes. NNMT expression in adipose tissue is increased in obesity and diabetes. Knockdown of NNMT prevents mice from developing diet-induced obesity, which is closely linked to insulin resistance. An early sign of systemic insulin resistance is reduced expression of glucose transporter 4 (GLUT4) selectively in adipose tissue. Adipose tissue-specific knockout and overexpression of GLUT4 cause reciprocal changes in NNMT expression. The aim of the current study was to elucidate the mechanism that regulates NNMT expression in adipocytes. METHODS: 3T3-L1 adipocytes were cultured in media with varying glucose concentrations or activators and inhibitors of intracellular pathways. NNMT mRNA and protein levels were measured with quantitative polymerase chain reaction and Western blotting. RESULTS: Glucose deprivation of 3T3-L1 adipocytes induced a 2-fold increase in NNMT mRNA and protein expression. This effect was mimicked by inhibition of glucose transport with phloretin, and by inhibition of glycolysis with the phosphoglucose isomerase inhibitor 2-deoxyglucose. Conversely, inhibition of the pentose phosphate pathway did not affect NNMT expression. Pharmacological activation of the cellular energy sensor AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin (mTOR) pathway caused an increase in NNMT levels that was similar to the effect of glucose deprivation. Activation of mTOR with MHY1485 prevented the effect of glucose deprivation on NNMT expression. Furthermore, upregulation of NNMT levels depended on functional autophagy and protein translation. CONCLUSION: Glucose availability regulates NNMT expression via an mTOR-dependent mechanism.


Assuntos
Adipócitos/efeitos dos fármacos , Transportador de Glucose Tipo 4/genética , Glucose/farmacologia , Nicotinamida N-Metiltransferase/genética , Serina-Treonina Quinases TOR/genética , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Transporte Biológico/efeitos dos fármacos , Diferenciação Celular , Desoxiglucose/farmacologia , Metabolismo Energético/genética , Regulação da Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 4/antagonistas & inibidores , Transportador de Glucose Tipo 4/metabolismo , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Homeostase/genética , Camundongos , Morfolinas/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Nicotinamida N-Metiltransferase/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/genética , Floretina/farmacologia , Biossíntese de Proteínas , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Triazinas/farmacologia
10.
Mol Carcinog ; 59(7): 691-700, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32115801

RESUMO

Triple-negative breast cancer (TNBC) lacks a well-defined molecular target and is associated with poorer outcomes compared to other breast cancer subtypes. Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade therapy shows a 10% to 20% response rate in TNBC patients. Our previous studies show that PD-L1 proteins are heavily glycosylated in TNBC, and the glycosylation plays an important role in the PD-L1 protein's stability and immunosuppressive function. However, a strategy for PD-L1 deglycosylation in TNBC is poorly defined. Here we found that a saccharide analog, 2-deoxy- d-glucose (2-DG), inhibits glycosylation of PD-L1 and its immunosuppressive function by combining with EGFR inhibitor, gefitinib. Interestingly, 2-DG/gefitinib-induced deglycosylation of PD-L1 decreased the expression level of PD-L1 protein as well as its binding with PD-1. However, there was no significant decrease in 4-1BB expression and its binding with 4-1BBL by 2-DG/gefitinib. Furthermore, we demonstrated that the combination treatment of 2-DG/gefitinib and 4-1BB antibody enhances antitumor immunity in TNBC syngeneic murine models. Together, our results suggest a new immunotherapeutic strategy to enhance antitumor immunity by PD-L1 deglycosylation and 4-1BB stimulation in TNBC.


Assuntos
Antineoplásicos/farmacologia , Antígeno B7-H1/metabolismo , Desoxiglucose/farmacologia , Glucose/farmacologia , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/terapia , Animais , Anticorpos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Gefitinibe/farmacologia , Células HEK293 , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C
11.
J Biol Chem ; 295(10): 3099-3114, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32005665

RESUMO

Upon activation with pathogen-associated molecular patterns, metabolism of macrophages and dendritic cells is shifted from oxidative phosphorylation to aerobic glycolysis, which is considered important for proinflammatory cytokine production. Fragments of bacterial peptidoglycan (muramyl peptides) activate innate immune cells through nucleotide-binding oligomerization domain (NOD) 1 and/or NOD2 receptors. Here, we show that NOD1 and NOD2 agonists induce early glycolytic reprogramming of human monocyte-derived macrophages (MDM), which is similar to that induced by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide. This glycolytic reprogramming depends on Akt kinases, independent of mTOR complex 1 and is efficiently inhibited by 2-deoxy-d-glucose (2-DG) or by glucose starvation. 2-DG inhibits proinflammatory cytokine production by MDM and monocyte-derived dendritic cells activated by NOD1 or TLR4 agonists, except for tumor necrosis factor production by MDM, which is inhibited initially, but augmented 4 h after addition of agonists and later. However, 2-DG exerts these effects by inducing unfolded protein response rather than by inhibiting glycolysis. By contrast, glucose starvation does not cause unfolded protein response and, in normoxic conditions, only marginally affects proinflammatory cytokine production triggered through NOD1 or TLR4. In hypoxia mimicked by treating MDM with oligomycin (a mitochondrial ATP synthase inhibitor), both 2-DG and glucose starvation strongly suppress tumor necrosis factor and interleukin-6 production and compromise cell viability. In summary, the requirement of glycolytic reprogramming for proinflammatory cytokine production in normoxia is not obvious, and effects of 2-DG on cytokine responses should be interpreted cautiously. In hypoxia, however, glycolysis becomes critical for cytokine production and cell survival.


Assuntos
Citocinas/metabolismo , Glicólise/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Proteína Adaptadora de Sinalização NOD1/agonistas , Receptor 4 Toll-Like/agonistas , Animais , Carboxiliases/metabolismo , Hipóxia Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Desoxiglucose/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Monócitos/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/metabolismo , Oligomicinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor 4 Toll-Like/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos
12.
J Immunol ; 204(5): 1373-1385, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31969386

RESUMO

Adoptive cellular therapy and its derivative, chimeric AgR T cell therapy, have achieved significant progress against cancer. Major barriers persist, however, including insufficient induction of cytotoxic T cells and exhaustion of tumor-infiltrating lymphocytes. In this study, we discovered a new role for 2-deoxy-d-glucose (2DG) in enhancing the antitumor activity of human T cells against NKG2D ligand-expressing tumor cells. Human T cells treated with 2DG upregulated the NK-specific transcription factors TOX2 and EOMES, thereby acquiring NK cell properties, including high levels of perforin/granzyme and increased sensitivity to IL-2. Notably, rather than inhibiting glycolysis, 2DG modified N-glycosylation, which augmented antitumor activity and cell surface retention of IL-2R of T cells. Moreover, 2DG treatment prevented T cells from binding to galectin-3, a potent tumor Ag associated with T cell anergy. Our results, therefore, suggest that modifying N-glycosylation of T cells with 2DG could improve the efficacy of T cell-based immunotherapies against cancer.


Assuntos
Desoxiglucose/farmacologia , Imunidade Celular/efeitos dos fármacos , Neoplasias/imunologia , Linfócitos T/imunologia , Glicosilação/efeitos dos fármacos , Proteínas HMGB/imunologia , Humanos , Imunoterapia , Interleucina-2/imunologia , Células K562 , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Proteínas com Domínio T/imunologia , Linfócitos T/patologia
13.
Int J Radiat Biol ; 96(5): 622-627, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31976790

RESUMO

Purpose: A study is presented of the irradiation of cancerous cervical cell line HeLa loaded with a platinum salt, betamethasone and deoxyglucose. The presence of the platinum increases the free-radical concentration and augments the cell death rate, whereas betamethasone or deoxyglucose induces radiosensitization by the alteration of metabolic pathways. Two by two combinations of these chemicals are made to investigate the possible benefit when two radiosensitizers are present. A model is proposed to understand the results of the presence of two modifying agents on the dose effects.Materials and methods: The cells were incubated for 6 h in the presence of the following molecules: dichloro terpyridine platinum, concentration C = 350 µM, betamethasone and deoxyglucose with concentrations of C = 0.2 µM and C = 6 mM, respectively. The cells were subsequently irradiated by carbon C6+ ion 290 MeV/amu up to a dose of 2.5 Gy, under atmospheric conditions.Results: The presence of the platinum salt or bethamethasone augments the cell death rate. The combination of betamethasone with the platinum salt also increases the cell death rate, but less than for the platinum salt alone. The explanation is that any radiosensitizer also behaves as a scavenger of free radicals. This dual behavior should be considered in any optimization of the design of radiosensitizers when different ionizing particles are used.


Assuntos
Radical Hidroxila , Terapia com Prótons , Radiossensibilizantes/farmacologia , Betametasona/farmacologia , Desoxiglucose/farmacologia , Células HeLa , Humanos , Transferência Linear de Energia , Modelos Teóricos , Compostos de Platina/farmacologia
14.
Oncol Rep ; 43(2): 711-717, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894283

RESUMO

The aim of the present study was to investigate the metabolic and anticancer effects of troglitazone (TGZ) with a focus on the potential role of mitochondrial pyruvate utilization. 2­Deoxyglucose (2­DG) was more cytotoxic in CT26 cancer cells compared with T47D cells, despite a smaller suppression of glucose uptake. On the other hand, TGZ caused a more prominent shift to glycolytic metabolism and was more cytotoxic in T47D cells. Both effects of TGZ on T47D cells were dose­dependently reversed by addition of methyl pyruvate (mPyr), indicating suppression of mitochondrial pyruvate availability. Furthermore, UK5099, a specific mitochondrial pyruvate carrier inhibitor, closely simulated the metabolic and antitumor effects of TGZ and their reversal by mPyr. This was accompanied by a substantial reduction of activated p70S6K. In CT26 cells, UK5099 did not reduce activated p70S6K and only modestly decreased cell proliferation. In these cells, combining glutamine restriction with UK5099 further increased glucose uptake and completely suppressed cell proliferation. Thus, TGZ­mediated inhibition of mitochondrial pyruvate utilization is an effective treatment for cancer cells that are more dependent on mitochondrial glucose metabolism. By contrast, cancer cells that are more glycolysis­dependent may require suppression of glutamine utilization in addition to blocking mitochondrial pyruvate availability for a full antitumor effect.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Mitocôndrias/metabolismo , Ácido Pirúvico/metabolismo , Troglitazona/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desoxiglucose/farmacologia , Relação Dose-Resposta a Droga , Feminino , Glicólise/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo
15.
Methods Mol Biol ; 2102: 3-15, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989547

RESUMO

Toxicology is a broad field that requires the translation of biochemical responses to xenobiotic exposures into useable information to ensure the safety of the public. Modern techniques are improving rapidly, both quantitatively and qualitatively, to provide the tools necessary to expand available toxicological datasets and refine our ability to translate that data into relevant information via bioinformatics. These new techniques can, and do, impact many of the current critical roles in toxicology, including the environmental, forensic, preclinical/clinical, and regulatory realms. One area of rapid expansion is our understanding of bioenergetics, or the study of the transformation of energy in living organisms, and new mathematical approaches are needed to interpret these large datasets. As bioenergetics are intimately involved in the regulation of how and when a cell responds to xenobiotics, monitoring these changes (i.e., metabolic fluctuations) in cells/tissues post-exposure provides an approach to define the temporal scale of pharmacodynamic responses, which can be used to guide additional toxicological techniques (e.g., "omics"). This chapter will summarize important in vitro assays and in vivo imaging techniques to take real-time measurements. Using this information, our laboratory has utilized bioenergetics to identify significant time points of pharmacodynamic relevance as well as forecast the cell's eventual fate.


Assuntos
Bioensaio/métodos , Metabolismo Energético/fisiologia , Mitocôndrias/metabolismo , Toxicologia/métodos , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , 4-Cloro-7-nitrobenzofurazano/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Desoxiglucose/análogos & derivados , Desoxiglucose/metabolismo , Desoxiglucose/farmacologia , Metabolismo Energético/efeitos dos fármacos , Fluordesoxiglucose F18/metabolismo , Humanos , Técnicas In Vitro , Verde de Indocianina/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , NAD/metabolismo , NADP/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Fluxo de Trabalho , Xenobióticos
16.
Br J Cancer ; 122(2): 209-220, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31819189

RESUMO

BACKGROUND: Mitochondrial dynamics plays an important role in tumour progression. However, how these dynamics integrate tumour metabolism in hepatocellular carcinoma (HCC) metastasis is still unclear. METHODS: The mitochondrial fusion protein mitofusin-1 (MFN1) expression and its prognostic value are detected in HCC. The effects and underlying mechanisms of MFN1 on HCC metastasis and metabolic reprogramming are analysed both in vitro and in vivo. RESULTS: Mitochondrial dynamics, represented by constant fission and fusion, are found to be associated with HCC metastasis. High metastatic HCC displays excessive mitochondrial fission. Among genes involved in mitochondrial dynamics, MFN1 is identified as a leading downregulated candidate that is closely associated with HCC metastasis and poor prognosis. While promoting mitochondrial fusion, MFN1 inhibits cell proliferation, invasion and migration capacity both in vitro and in vivo. Mechanistically, disruption of mitochondrial dynamics by depletion of MFN1 triggers the epithelial-to-mesenchymal transition (EMT) of HCC. Moreover, MFN1 modulates HCC metastasis by metabolic shift from aerobic glycolysis to oxidative phosphorylation. Treatment with glycolytic inhibitor 2-Deoxy-D-glucose (2-DG) significantly suppresses the effects induced by depletion of MFN1. CONCLUSIONS: Our results reveal a critical involvement of mitochondrial dynamics in HCC metastasis via modulating glucose metabolic reprogramming. MFN1 may serve as a novel potential therapeutic target for HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Desoxiglucose/farmacologia , GTP Fosfo-Hidrolases/genética , Glucose/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , GTP Fosfo-Hidrolases/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Metástase Neoplásica , Fosforilação Oxidativa/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Biosci Biotechnol Biochem ; 84(1): 198-207, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31566090

RESUMO

High glycosidase-producing strains of Aspergillus luchuensis were isolated from 2-deoxyglucose (2-DG) resistant mutants. α-Amylase, exo-α-1,4-glucosidase, ß-glucosidase and ß-xylosidase activity in the mutants was ~3, ~2, ~4 and ~2.5 times higher than the parental strain RIB2604 on koji-making conditions, respectively. Citric acid production and mycelia growth of the mutants, however, approximately halved to that of the parent. Compared to the parent, the alcohol yield from rice and sweet potato shochu mash of the mutant increased ~5.7% and 3.0%, respectively. The mutant strains showed significantly low glucose assimilability despite the fructose one was almost normal, and they had a single missense or nonsense mutation in the glucokinase gene glkA. The recombinant strain that was introduced at one of the mutations, glkA Q300K, demonstrated similar but not identical phenotypes to the mutant strain. This result indicates that glkA Q300K is one of the major mutations in 2-DG resistant strains.


Assuntos
Aspergillus/genética , Aspergillus/isolamento & purificação , Separação Celular/métodos , Códon sem Sentido/genética , Genes Fúngicos/genética , alfa-Glucosidases/metabolismo , Aspergillus/classificação , Aspergillus/metabolismo , Catepsina A/metabolismo , Ácido Cítrico/metabolismo , Desoxiglucose/farmacologia , Farmacorresistência Fúngica , Etanol/metabolismo , Fermentação , Alimentos e Bebidas Fermentados/microbiologia , Frutose/metabolismo , Glucoquinase/genética , Glucose/metabolismo , Ipomoea batatas/química , Oryza/química , Fenótipo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Xilosidases/metabolismo , alfa-Amilases/metabolismo , beta-Glucosidase/metabolismo
18.
Food Chem ; 311: 125905, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31796226

RESUMO

The antibacterial activities of the dicarbonyl compounds glyoxal (GO), methylglyoxal (MGO), 3-deoxyglucosone (3-DG) were assessed against Gram-positive and Gram-negative pathogenic and food spoilage bacteria, both in agarised and liquid assay system. The kinetics of dicarbonyls' degradation at different antimicrobial assay conditions were studied, to determine the possible interference of the nutrient medium. In agarised assay system, GO and MGO exhibited antimicrobial activity, with higher efficacy against Gram-positive strains than Gram-negative ones. The nutrient medium reacted quickly both with GO and MGO, interfering with the antibacterial potential and the degradation kinetics indicated first-order reactions. In liquid assay system, both GO and MGO inhibited the target bacteria at concentrations significantly lower than those estimated in agarised assay system. Moreover, to the best of our knowledge, the antibacterial activity of GO and MGO against Listeria innocua, Pseudomonas fluorescens, Salmonella enterica and Bacillus cereus has not been previously reported.


Assuntos
Antibacterianos/farmacologia , Desoxiglucose/análogos & derivados , Glioxal/farmacologia , Aldeído Pirúvico/farmacologia , Antibacterianos/química , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/crescimento & desenvolvimento , Desoxiglucose/química , Desoxiglucose/farmacologia , Glioxal/química , Cinética , Testes de Sensibilidade Microbiana , Pseudomonas fluorescens/efeitos dos fármacos , Pseudomonas fluorescens/crescimento & desenvolvimento , Aldeído Pirúvico/química , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/crescimento & desenvolvimento
19.
PLoS One ; 14(11): e0224405, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31710612

RESUMO

A subset of environmental chemicals acts as "obesogens" as they increase adipose mass and lipid content in livers of treated rodents. One of the most studied class of obesogens are the tin-containing chemicals that have as a central moiety tributyltin (TBT), which bind and activate two nuclear hormone receptors, Peroxisome Proliferator Activated Receptor Gamma (PPARG) and Retinoid X Receptor Alpha (RXRA), at nanomolar concentrations. Here, we have tested whether TBT chloride at such concentrations may affect the neutral lipid level in two cell line models of human liver. Indeed, using high content image analysis (HCA), TBT significantly increased neutral lipid content in a time- and concentration-dependent manner. Consistent with the observed increased lipid accumulation, RNA fluorescence in situ hybridization (RNA FISH) and RT-qPCR experiments revealed that TBT enhanced the steady-state mRNA levels of two key genes for de novo lipogenesis, the transcription factor SREBF1 and its downstream enzymatic target, FASN. Importantly, pre-treatment of cells with 2-deoxy-D-glucose reduced TBT-mediated lipid accumulation, thereby suggesting a role for active glycolysis during the process of lipid accumulation. As other RXRA binding ligands can promote RXRA protein turnover via the 26S proteasome, TBT was tested for such an effect in the two liver cell lines. We found that TBT, in a time- and dose-dependent manner, significantly reduced steady-state RXRA levels in a proteasome-dependent manner. While TBT promotes both RXRA protein turnover and lipid accumulation, we found no correlation between these two events at the single cell level, thereby suggesting an additional mechanism may be involved in TBT promotion of lipid accumulation, such as glycolysis.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Receptor X Retinoide alfa/metabolismo , Compostos de Trialquitina/farmacologia , Linhagem Celular , Desoxiglucose/farmacologia , Relação Dose-Resposta a Droga , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Hibridização in Situ Fluorescente , Receptor X Retinoide alfa/genética
20.
Nat Commun ; 10(1): 5091, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31704924

RESUMO

Activated macrophages switch from oxidative phosphorylation to aerobic glycolysis, similar to the Warburg effect, presenting a potential therapeutic target in inflammatory disease. The endogenous metabolite itaconate has been reported to regulate macrophage function, but its precise mechanism is not clear. Here, we show that 4-octyl itaconate (4-OI, a cell-permeable itaconate derivative) directly alkylates cysteine residue 22 on the glycolytic enzyme GAPDH and decreases its enzyme activity. Glycolytic flux analysis by U13C glucose tracing provides evidence that 4-OI blocks glycolytic flux at GAPDH. 4-OI thereby downregulates aerobic glycolysis in activated macrophages, which is required for its anti-inflammatory effects. The anti-inflammatory effects of 4-OI are replicated by heptelidic acid, 2-DG and reversed by increasing wild-type (but not C22A mutant) GAPDH expression. 4-OI protects against lipopolysaccharide-induced lethality in vivo and inhibits cytokine release. These findings show that 4-OI has anti-inflammatory effects by targeting GAPDH to decrease aerobic glycolysis in macrophages.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Succinatos/farmacologia , Alquilação , Animais , Antimetabólitos/farmacologia , Cisteína/efeitos dos fármacos , Cisteína/genética , Cisteína/metabolismo , Desoxiglucose/farmacologia , Regulação para Baixo , Endotoxemia/imunologia , Glucose/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/antagonistas & inibidores , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Inflamação/imunologia , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/imunologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/imunologia , Fosforilação Oxidativa/efeitos dos fármacos , Células RAW 264.7 , Sesquiterpenos/farmacologia
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