RESUMO
Metabolic reprogramming is a key feature of many cancers, but how and when it contributes to tumorigenesis remains unclear. Here we demonstrate that metabolic reprogramming induced by mitochondrial fusion can be rate-limiting for immortalization of tumor-initiating cells (TICs) and trigger their irreversible dedication to tumorigenesis. Using single-cell transcriptomics, we find that Drosophila brain tumors contain a rapidly dividing stem cell population defined by upregulation of oxidative phosphorylation (OxPhos). We combine targeted metabolomics and in vivo genetic screening to demonstrate that OxPhos is required for tumor cell immortalization but dispensable in neural stem cells (NSCs) giving rise to tumors. Employing an in vivo NADH/NAD+ sensor, we show that NSCs precisely increase OxPhos during immortalization. Blocking OxPhos or mitochondrial fusion stalls TICs in quiescence and prevents tumorigenesis through impaired NAD+ regeneration. Our work establishes a unique connection between cellular metabolism and immortalization of tumor-initiating cells.
Assuntos
Neoplasias Encefálicas/metabolismo , Carcinogênese/metabolismo , Transformação Celular Neoplásica/metabolismo , Dinâmica Mitocondrial , NAD/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/metabolismo , Fosforilação Oxidativa , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Carcinogênese/genética , Carcinogênese/patologia , Transformação Celular Neoplásica/patologia , Ciclo do Ácido Cítrico/genética , Biologia Computacional , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Glicólise/genética , Espectrometria de Massas , Metabolômica , Microscopia Eletrônica de Transmissão , Família Multigênica , Células-Tronco Neurais/patologia , Consumo de Oxigênio/genética , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Análise de Célula Única , Transcriptoma/genéticaRESUMO
There is considerable inter-individual variability in susceptibility to weight gain despite an equally obesogenic environment in large parts of the world. Whereas many studies have focused on identifying the genetic susceptibility to obesity, we performed a GWAS on metabolically healthy thin individuals (lowest 6th percentile of the population-wide BMI spectrum) in a uniquely phenotyped Estonian cohort. We discovered anaplastic lymphoma kinase (ALK) as a candidate thinness gene. In Drosophila, RNAi mediated knockdown of Alk led to decreased triglyceride levels. In mice, genetic deletion of Alk resulted in thin animals with marked resistance to diet- and leptin-mutation-induced obesity. Mechanistically, we found that ALK expression in hypothalamic neurons controls energy expenditure via sympathetic control of adipose tissue lipolysis. Our genetic and mechanistic experiments identify ALK as a thinness gene, which is involved in the resistance to weight gain.
Assuntos
Quinase do Linfoma Anaplásico/genética , Magreza/genética , Tecido Adiposo/metabolismo , Adulto , Animais , Linhagem Celular , Estudos de Coortes , Drosophila/genética , Estônia , Feminino , Humanos , Leptina/genética , Lipólise/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Interferência de RNA/fisiologia , Adulto JovemRESUMO
Stem cells are highly abundant during early development but become a rare population in most adult organs. The molecular mechanisms causing stem cells to exit proliferation at a specific time are not well understood. Here, we show that changes in energy metabolism induced by the steroid hormone ecdysone and the Mediator initiate an irreversible cascade of events leading to cell-cycle exit in Drosophila neural stem cells. We show that the timely induction of oxidative phosphorylation and the mitochondrial respiratory chain are required in neuroblasts to uncouple the cell cycle from cell growth. This results in a progressive reduction in neuroblast cell size and ultimately in terminal differentiation. Brain tumor mutant neuroblasts fail to undergo this shrinkage process and continue to proliferate until adulthood. Our findings show that cell size control can be modified by systemic hormonal signaling and reveal a unique connection between metabolism and proliferation in stem cells.
Assuntos
Proliferação de Células , Drosophila melanogaster/citologia , Ecdisona/metabolismo , Células-Tronco Neurais/citologia , Animais , Tamanho Celular , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Metabolismo Energético , Genoma de Inseto , Complexo Mediador/metabolismo , Células-Tronco Neurais/metabolismoRESUMO
Obesity and diabetes affect more than half a billion individuals worldwide. Interestingly, the two conditions do not always coincide and the molecular determinants of "healthy" versus "unhealthy" obesity remain ill-defined. Chronic metabolic inflammation (metaflammation) is believed to be pivotal. Here, we tested a hypothesized anti-inflammatory role for heme oxygenase-1 (HO-1) in the development of metabolic disease. Surprisingly, in matched biopsies from "healthy" versus insulin-resistant obese subjects we find HO-1 to be among the strongest positive predictors of metabolic disease in humans. We find that hepatocyte and macrophage conditional HO-1 deletion in mice evokes resistance to diet-induced insulin resistance and inflammation, dramatically reducing secondary disease such as steatosis and liver toxicity. Intriguingly, cellular assays show that HO-1 defines prestimulation thresholds for inflammatory skewing and NF-κB amplification in macrophages and for insulin signaling in hepatocytes. These findings identify HO-1 inhibition as a potential therapeutic strategy for metabolic disease.
Assuntos
Heme Oxigenase-1/metabolismo , Resistência à Insulina , Proteínas de Membrana/metabolismo , Obesidade/complicações , Tecido Adiposo/metabolismo , Animais , Dieta Hiperlipídica , Hepatócitos/metabolismo , Humanos , Inflamação/metabolismo , Fígado/metabolismo , Macrófagos/metabolismo , Doenças Metabólicas/metabolismo , Doenças Metabólicas/fisiopatologia , Camundongos , Camundongos Knockout , Obesidade/fisiopatologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.
Assuntos
Infecções por Bactérias Gram-Negativas/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Heme/metabolismo , Hemólise/imunologia , Macrófagos/imunologia , Fagocitose , Sepse/imunologia , Animais , Antibacterianos/uso terapêutico , Citoesqueleto/metabolismo , Feminino , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Fatores de Troca do Nucleotídeo Guanina/genética , Heme Oxigenase-1/genética , Hemólise/efeitos dos fármacos , Humanos , Evasão da Resposta Imune , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/efeitos dos fármacos , Quinina/uso terapêutico , Células RAW 264.7 , Sepse/tratamento farmacológico , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Diabetes, obesity, and cancer affect upward of 15% of the world's population. Interestingly, all three diseases juxtapose dysregulated intracellular signaling with altered metabolic state. Exactly which genetic factors define stable metabolic set points in vivo remains poorly understood. Here, we show that hedgehog signaling rewires cellular metabolism. We identify a cilium-dependent Smo-Ca(2+)-Ampk axis that triggers rapid Warburg-like metabolic reprogramming within minutes of activation and is required for proper metabolic selectivity and flexibility. We show that Smo modulators can uncouple the Smo-Ampk axis from canonical signaling and identify cyclopamine as one of a new class of "selective partial agonists," capable of concomitant inhibition of canonical and activation of noncanonical hedgehog signaling. Intriguingly, activation of the Smo-Ampk axis in vivo drives robust insulin-independent glucose uptake in muscle and brown adipose tissue. These data identify multiple noncanonical endpoints that are pivotal for rational design of hedgehog modulators and provide a new therapeutic avenue for obesity and diabetes.
Assuntos
Tecido Adiposo Marrom/metabolismo , Glicólise , Proteínas Hedgehog/metabolismo , Células Musculares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Quinases Proteína-Quinases Ativadas por AMP , Adipócitos/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Cílios/metabolismo , Diabetes Mellitus/metabolismo , Humanos , Camundongos , Neoplasias/metabolismo , Obesidade/metabolismo , Proteínas Quinases/metabolismo , Receptor SmoothenedRESUMO
ABSTRACT: Systemic mastocytosis (SM) is defined by the expansion and accumulation of neoplastic mast cells (MCs) in the bone marrow (BM) and extracutaneous organs. Most patients harbor a somatic KIT D816V mutation, which leads to growth factor-independent KIT activation and accumulation of MC. Tumor necrosis factor α (TNF) is a proapoptotic and inflammatory cytokine that has been implicated in the clonal selection of neoplastic cells. We found that KIT D816V increases the expression and secretion of TNF. TNF expression in neoplastic MCs is reduced by KIT-targeting drugs. Similarly, knockdown of KIT or targeting the downstream signaling cascade of MAPK and NF-κB signaling reduced TNF expression levels. TNF reduces colony formation in human BM cells, whereas KIT D816V+ cells are less susceptible to the cytokine, potentially contributing to clonal selection. In line, knockout of TNF in neoplastic MC prolonged survival and reduced myelosuppression in a murine xenotransplantation model. Mechanistic studies revealed that the relative resistance of KIT D816V+ cells to TNF is mediated by the apoptosis-regulator BIRC5 (survivin). Expression of BIRC5 in neoplastic MC was confirmed by immunohistochemistry of samples from patients with SM. TNF serum levels are significantly elevated in patients with SM and high TNF levels were identified as a biomarker associated with inferior survival. We here characterized TNF as a KIT D816V-dependent cytokine that promotes clonal dominance. We propose TNF and apoptosis-associated proteins as potential therapeutic targets in SM.
Assuntos
Mastocitose Sistêmica , Mastocitose , Humanos , Animais , Camundongos , Fator de Necrose Tumoral alfa , Survivina/genética , Prognóstico , Mastocitose Sistêmica/diagnóstico , Mastocitose Sistêmica/genética , CitocinasRESUMO
Over 1 billion people are estimated to be overweight, placing them at risk for diabetes, cardiovascular disease, and cancer. We performed a systems-level genetic dissection of adiposity regulation using genome-wide RNAi screening in adult Drosophila. As a follow-up, the resulting approximately 500 candidate obesity genes were functionally classified using muscle-, oenocyte-, fat-body-, and neuronal-specific knockdown in vivo and revealed hedgehog signaling as the top-scoring fat-body-specific pathway. To extrapolate these findings into mammals, we generated fat-specific hedgehog-activation mutant mice. Intriguingly, these mice displayed near total loss of white, but not brown, fat compartments. Mechanistically, activation of hedgehog signaling irreversibly blocked differentiation of white adipocytes through direct, coordinate modulation of early adipogenic factors. These findings identify a role for hedgehog signaling in white/brown adipocyte determination and link in vivo RNAi-based scanning of the Drosophila genome to regulation of adipocyte cell fate in mammals.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Obesidade/genética , Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Adipogenia , Animais , AMP Cíclico/metabolismo , Glucocorticoides/metabolismo , Humanos , Camundongos , Camundongos Knockout , Células Musculares/metabolismo , Proteínas Repressoras/genéticaRESUMO
Mastocytosis is a hematopoietic neoplasm characterized by expansion of KIT D816V-mutated clonal mast cells in various organs and severe or even life-threatening anaphylactic reactions. Recently, hereditary α-tryptasemia (HαT) has been described as a common genetic trait with increased copy numbers of the α-tryptase encoding gene, TPSAB1, and associated with an increased basal serum tryptase level and a risk of mast cell activation. The purpose of our study was to elucidate the clinical relevance of HαT in patients with mastocytosis. TPSAB1 germline copy number variants were assessed by digital polymerase chain reaction in 180 mastocytosis patients, 180 sex-matched control subjects, 720 patients with other myeloid neoplasms, and 61 additional mastocytosis patients of an independent validation cohort. α-Tryptase encoding TPSAB1 copy number gains, compatible with HαT, were identified in 17.2% of mastocytosis patients and 4.4% of the control population (P < .001). Patients with HαT exhibited higher tryptase levels than patients without HαT (median tryptase in HαT+ cases: 49.6 ng/mL vs HαT- cases: 34.5 ng/mL, P = .004) independent of the mast cell burden. Hymenoptera venom hypersensitivity reactions and severe cardiovascular mediator-related symptoms/anaphylaxis were by far more frequently observed in mastocytosis patients with HαT than in those without HαT. Results were confirmed in an independent validation cohort. The high prevalence of HαT in mastocytosis hints at a potential pathogenic role of germline α-tryptase encoding TPSAB1 copy number gains in disease evolution. Together, our data suggest that HαT is a novel emerging robust biomarker in mastocytosis that is useful for determining the individual patient´s risk of developing severe anaphylaxis.
Assuntos
Mastocitose , Triptases/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Variações do Número de Cópias de DNA , Feminino , Marcadores Genéticos , Humanos , Masculino , Mastocitose/sangue , Mastocitose/genética , Pessoa de Meia-Idade , Triptases/sangue , Adulto JovemRESUMO
While intrinsic changes in aging hematopoietic stem cells (HSCs) are well characterized, it remains unclear how extrinsic factors affect HSC aging. Here, we demonstrate that cells in the niche-endothelial cells (ECs) and CXCL12-abundant reticular cells (CARs)-highly express the heme-degrading enzyme, heme oxygenase 1 (HO-1), but then decrease its expression with age. HO-1-deficient animals (HO-1-/- ) have altered numbers of ECs and CARs that produce less hematopoietic factors. HSCs co-cultured in vitro with HO-1-/- mesenchymal stromal cells expand, but have altered kinetic of growth and differentiation of derived colonies. HSCs from young HO-1-/- animals have reduced quiescence and regenerative potential. Young HO-1-/- HSCs exhibit features of premature exhaustion on the transcriptional and functional level. HO-1+/+ HSCs transplanted into HO-1-/- recipients exhaust their regenerative potential early and do not reconstitute secondary recipients. In turn, transplantation of HO-1-/- HSCs to the HO-1+/+ recipients recovers the regenerative potential of HO-1-/- HSCs and reverses their transcriptional alterations. Thus, HSC-extrinsic activity of HO-1 prevents HSCs from premature exhaustion and may restore the function of aged HSCs.
Assuntos
Heme Oxigenase-1 , Células-Tronco Mesenquimais , Animais , Diferenciação Celular , Células Endoteliais , Células-Tronco Hematopoéticas , Heme Oxigenase-1/genéticaRESUMO
A high allele burden of the KIT D816V mutation in peripheral blood or bone marrow aspirates indicates multi-lineage hematopoietic involvement and has been associated with an aggressive clinical course of systemic mastocytosis. Since mast cells are substantially underrepresented in these liquid specimens, their mutation burden likely underestimates the tumor burden of the disease. We used a novel previously validated digital polymerase chain reaction (PCR) method for KIT D816V analysis to systematically analyze the mutation burden in formalin-fixed, paraffin-embedded bone marrow tissue sections of 116 mastocytosis patients (91 with indolent and 25 with advanced systemic mastocytosis), and to evaluate for the first time the clinical value of the tissue mutation burden as a novel biomarker. The KIT D816V mutation burden in the tissue was significantly higher and correlated better with bone marrow mast cell infiltration (r=0.68 vs 0.48) and serum tryptase levels (r=0.68 vs 0.58) compared to that in liquid specimens. Furthermore, the KIT D816V tissue mutation burden was: (i) significantly higher in advanced than in indolent systemic mastocytosis (P=0.001); (ii) predicted survival of patients in multivariate analyses independently; and (iii) was significantly reduced after response to cytoreductive therapy. Finally, digital PCR was more sensitive in detecting KIT D816V in bone marrow sections of indolent systemic mastocytosis patients than melting curve analysis after peptide nucleic acid-mediated PCR clamping (97% vs 89%; P<0.05). In summary, digital PCR-based measurement of KIT D816V mutation burden in the tissue represents a novel biomarker with independent prognostic significance that can also be employed for monitoring disease progression and treatment response in systemic mastocytosis.
Assuntos
Mastocitose Sistêmica , Mastocitose , Biomarcadores , Humanos , Mastócitos , Mastocitose/diagnóstico , Mastocitose/genética , Mastocitose Sistêmica/diagnóstico , Mastocitose Sistêmica/genética , Mutação , Proteínas Proto-Oncogênicas c-kit/genéticaRESUMO
Methods to estimate bone marrow plasma cells (BMPC) basically include histopathology, cytomorphology, and flow cytometry. The present study compares the outcomes of these methods with special focus on the impact of BMPC-specific characteristics on their recovery by either method. Laboratory reports of diagnostic samples from 238 consecutive patients with suspected or known plasma cell disease were retrospectively analyzed. The median (IQR) proportion of BMPC was 30.0% (15.0-70.0%) by histological review (hBMPC), 7.0% (2.0-16.0%) by smear review (sBMPC), and 3.0% (0.8-10.0%) by flow cytometry (fBMPC). The disparity of results between core biopsy and aspirate smear was enhanced in case of poor quality of the smear, increased BM fiber content, higher grade cell atypia, expression of CD56 (all P < 0.0001), the number of cytogenetic aberrations (P = 0.0002), and abnormalities of the MYC gene (P = 0.0002). Conversely, expression of CD19 and a non-clonal plasma cell phenotype were associated with a lower difference between hBMPC and sBMPC (both P < 0.0001). The disparity between the percentages of sBMPC and fBMPC was associated with the quality of the smear (P = 0.0007) and expression of CD56 (P < 0.0001). Our results suggest that the recovery of BMPC in aspirate specimens not only is a matter of sampling quality but also depends on biological cell properties. Aspiration failure due to malignant type features of BMPC may lead to misclassification of plasma cell disorders and represent a bias for the detection of minimal residual disease after therapy.
Assuntos
Antígenos CD19/biossíntese , Células da Medula Óssea , Antígeno CD56/biossíntese , Mieloma Múltiplo , Proteínas de Neoplasias/biossíntese , Plasmócitos , Adulto , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/classificação , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Neoplasia Residual , Plasmócitos/metabolismo , Plasmócitos/patologia , Estudos RetrospectivosRESUMO
Background Monitoring of molecular response (MR) using quantitative polymerase chain reaction (PCR) for BCR-ABL1 is a pivotal tool for guiding tyrosine kinase inhibitor therapy and the long-term follow-up of patients with chronic myeloid leukemia (CML). Results of MR monitoring are standardized according to the International Scale (IS), and specific time-dependent molecular milestones for definition of optimal response and treatment failure have been included in treatment recommendations. The common practice to use peripheral blood (PB) instead of bone marrow (BM) aspirate to monitor the MR monitoring in CML has been questioned. Some studies described differences between BCR-ABL1 levels in paired PB and BM specimens. Methods We examined 631 paired PB and BM samples from 283 CML patients in a retrospective single-center study using an IS normalized quantitative reverse transcription (qRT)-PCR assay for quantification of BCR-ABL1IS. Results A good overall concordance of BCR-ABL1IS results was found, a systematic tendency towards higher BCR-ABL1IS levels in PB was observed in samples of CML patients in a major MR. This difference was most pronounced in patients treated with imatinib for at least 1 year. Importantly, the difference resulted in a significantly lower rate of deep MR when BCR-ABL1IS was assessed in the PB compared to BM aspirates. Conclusions In summary, our data suggest that the classification of deep MR in patients with CML is more stringent in PB than in BM. Our study supports the current practice to primarily use PB for long-term molecular follow-up monitoring in CML.
Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Terapia de Alvo Molecular/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Medula Óssea/patologia , Feminino , Proteínas de Fusão bcr-abl/sangue , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
OBJECTIVE: Liver injury impacts hepatic inflammation in part via Toll-like receptor (TLR) signalling. Triggering receptor expressed on myeloid cells 2 (TREM-2) modulates TLR4-mediated inflammation in bone marrow (BM)-derived macrophages but its function in liver injury is unknown. Here we hypothesised that the anti-inflammatory effects of TREM-2 on TLR signalling may limit hepatic injury. DESIGN: TREM-2 expression was analysed in livers of humans with various forms of liver injury compared with control individuals. Acute and chronic liver injury models were performed in wild type and Trem-2-/- mice. Primary liver cells from both genotypes of mice were isolated for in vitro experiments. RESULTS: TREM-2 was expressed on non-parenchymal hepatic cells and induced during liver injury in mice and man. Mice lacking TREM-2 exhibited heightened liver damage and inflammation during acute and repetitive carbon tetrachloride and acetaminophen (APAP) intoxication, the latter of which TREM-2 deficiency was remarkably associated with worsened survival. Liver damage in Trem-2-/- mice following chronic injury and APAP challenge was associated with elevated hepatic lipid peroxidation and macrophage content. BM transplantation experiments and cellular reactive oxygen species assays revealed effects of TREM-2 in the context of chronic injury depended on both immune and resident TREM-2 expression. Consistent with effects of TREM-2 on inflammation-associated injury, primary hepatic macrophages and hepatic stellate cells lacking TREM-2 exhibited augmented TLR4-driven proinflammatory responses. CONCLUSION: Our data indicate that by acting as a natural brake on inflammation during hepatocellular injury, TREM-2 is a critical regulator of diverse types of hepatotoxic injury.
Assuntos
Cirrose Hepática/metabolismo , Fígado/metabolismo , Glicoproteínas de Membrana/fisiologia , Receptores Imunológicos/fisiologia , Acetaminofen , Idoso , Animais , Tetracloreto de Carbono , Estudos de Casos e Controles , Feminino , Células-Tronco Hematopoéticas/metabolismo , Hepatócitos/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Células de Kupffer/metabolismo , Peroxidação de Lipídeos/fisiologia , Cirrose Hepática/etiologia , Cirrose Hepática/imunologia , Cirrose Hepática Experimental/imunologia , Cirrose Hepática Experimental/metabolismo , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos Knockout , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/metabolismo , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptor 4 Toll-Like/fisiologia , Regulação para Cima/fisiologiaRESUMO
Macrophages are central for the immune control of intracellular microbes. Heme oxygenase 1 (HO-1, hmox) is the first and rate limiting enzyme in the breakdown of heme originating from degraded senescent erythrocytes and heme-proteins, yielding equal amounts of iron, carbon monoxide and biliverdin. HO-1 is strongly up-regulated in macrophages in response to inflammatory signals, including bacterial endotoxin. In view of the essential role of iron for the growth and proliferation of intracellular bacteria along with known effects of the metal on innate immune function, we examined whether HO-1 plays a role in the control of infection with the intracellular bacterium Salmonella Typhimurium. We studied the course of infection in stably-transfected murine macrophages (RAW264.7) bearing a tetracycline-inducible plasmid producing hmox shRNA and in primary HO-1 knockout macrophages. While uptake of bacteria into macrophages was not affected, a significantly reduced survival of intracellular Salmonella was observed upon hmox knockdown or pharmacological hmox inhibition, which was independent of Nramp1 functionality. This could be traced to limitation of iron availability for intramacrophage bacteria along with enhanced stimulation of innate immune effector pathways, including the formation of reactive oxygen and nitrogen species and increased TNF-α expression. Mechanistically, these latter effects result from intracellular iron limitation with subsequent activation of NF-κB and further inos, tnfa and p47phox transcription along with reduced formation of the anti-inflammatory and radical scavenging molecules, CO and biliverdin as a consequence of HO-1 silencing. Taken together our data provide novel evidence that the infection-driven induction of HO-1 exerts detrimental effects in the early control of Salmonella infection, whereas hmox inhibition can favourably modulate anti-bacterial immune effector pathways of macrophages and promote bacterial elimination.
Assuntos
Heme Oxigenase-1/fisiologia , Proteínas de Membrana/fisiologia , Infecções por Salmonella/enzimologia , Salmonella typhimurium/imunologia , Animais , Indução Enzimática , Expressão Gênica/imunologia , Células HEK293 , Humanos , Imunidade Inata , Ferro/metabolismo , Camundongos , Viabilidade Microbiana , NF-kappa B/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Infecções por Salmonella/microbiologiaRESUMO
Obesity and diabetes represent key healthcare challenges of our day, affecting upwards of one billion people worldwide. These individuals are at higher risk for cancer, stroke, blindness, heart and cardiovascular disease, and to date, have no effective long-term treatment options available. Recent and accumulating evidence has implicated the developmental morphogen Hedgehog and its downstream signalling in metabolic control. Generally thought to be quiescent in adults, Hedgehog is associated with several human cancers, and as such, has already emerged as a therapeutic target in oncology. Here, we attempt to give a comprehensive overview of the key signalling events associated with both canonical and non-canonical Hedgehog signalling, and highlight the increasingly complex regulatory modalities that appear to link Hedgehog and control metabolism. We highlight these key findings and discuss their impact for therapeutic development, cancer and metabolic disease.
Assuntos
Proteínas Hedgehog/fisiologia , Transdução de Sinais , Tecido Adiposo/metabolismo , Animais , Metabolismo Energético , Humanos , Fígado/metabolismo , Músculo Esquelético/metabolismo , Neoplasias/metabolismo , Pâncreas/metabolismoRESUMO
UNLABELLED: Growth hormone (GH) resistance has been associated with liver cirrhosis in humans but its contribution to the disease remains controversial. In order to elucidate whether GH resistance plays a causal role in the establishment and development of liver fibrosis, or rather represents a major consequence thereof, we challenged mice lacking the GH receptor gene (Ghr(-/-), a model for GH resistance) by crossing them with Mdr2 knockout mice (Mdr2(-/-)), a mouse model of inflammatory cholestasis and liver fibrosis. Ghr(-/-);Mdr2(-/-) mice showed elevated serum markers associated with liver damage and cholestasis, extensive bile duct proliferation, and increased collagen deposition relative to Mdr2(-/-) mice, thus suggesting a more severe liver fibrosis phenotype. Additionally, Ghr(-/-);Mdr2(-/-) mice had a pronounced down-regulation of hepatoprotective genes Hnf6, Egfr, and Igf-1, and significantly increased levels of reactive oxygen species (ROS) and apoptosis in hepatocytes, compared to control mice. Moreover, single knockout mice (Ghr(-/-)) fed with a diet containing 1% cholic acid displayed an increase in hepatocyte ROS production, hepatocyte apoptosis, and bile infarcts compared to their wild-type littermates, indicating that loss of Ghr renders hepatocytes more susceptible to toxic bile acid accumulation. Surprisingly, and despite their severe fibrotic phenotype, Ghr(-/-);Mdr2(-/-) mice displayed a significant decrease in tumor incidence compared to Mdr2(-/-) mice, indicating that loss of Ghr signaling may slow the progression from fibrosis/cirrhosis to cancer in the liver. CONCLUSION: GH resistance dramatically exacerbates liver fibrosis in a mouse model of inflammatory cholestasis, therefore suggesting that GH resistance plays a causal role in the disease and provides a novel target for the development of liver fibrosis treatments.
Assuntos
Hormônio do Crescimento/metabolismo , Cirrose Hepática/etiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Ácidos e Sais Biliares/metabolismo , Colestase/complicações , Hepatócitos/fisiologia , Homeostase , Cirrose Hepática/metabolismo , Neoplasias Hepáticas Experimentais/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Receptores da Somatotropina/genética , Regulação para Cima , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
BACKGROUND: Infiltration of white adipose tissue (WAT) by inflammatory cells in obesity is considered to be a key event in the development of insulin resistance. Recently, mast cells (MCs) have been identified as new players in the pathogenesis of obesity. We aimed to investigate the relationship between MCs and various inflammatory markers in serum and WAT and to determine the role of MCs in the aetiology of insulin resistance. MATERIALS AND METHODS: Gene expression was measured in WAT from 20 morbidly obese patients and 20 nonobese control subjects. Homoeostasis Model of Assessment-Insulin Resistance (HOMA-IR) was used to estimate insulin sensitivity. In addition, wild-type and mast cell-deficient mice were fed a high-fat or low-fat diet to study mast cell influence on inflammatory cell polarization in WAT and overall metabolic changes. RESULTS: WAT levels of MC-specific TPSb2 transcript were increased in obesity and significantly positively correlated with TNF, CCL2, CCL5 and CD68 gene expression levels in our study subjects after adjustment for sex, age and BMI. Accordingly, MC deficiency abrogated increase in expression of pro-inflammatory M1 macrophage marker genes in mouse WAT upon high-fat diet feeding. However, MCs accumulated in obese human WAT independent of insulin resistance and systemic changes in inflammatory mediators. CONCLUSIONS: Our results suggest that MCs contribute to the local pro-inflammatory state within WAT in obesity but do not play a primary role in causing insulin resistance.
Assuntos
Resistência à Insulina/fisiologia , Mastócitos/fisiologia , Obesidade Mórbida/patologia , Tecido Adiposo Branco/patologia , Adulto , Animais , Biomarcadores/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Citocinas/metabolismo , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Fenótipo , Magreza/patologia , Triptases/metabolismoRESUMO
Hippocampal volume loss has been related to chronic stress as well as genetic factors. Although genetic and environmental variables affecting hippocampal volume have extensively been studied and related to mental illness, limited evidence is available with respect to G × E interactions on hippocampal volume. The present MRI study investigated interaction effects on hippocampal volume between three well-studied functional genetic variants (COMT Val158Met, BDNF Val66Met, 5-HTTLPR) associated with hippocampal volume and a measure of environmental adversity (life events questionnaire) in a large sample of healthy humans (n = 153). All three variants showed significant interactions with environmental adversity with respect to hippocampal volume. Observed effects were additive by nature and driven by both recent as well as early life events. A consecutive analysis of hippocampal subfields revealed a spatially distinct profile for each genetic variant suggesting a specific role of 5-HTTLPR for the subiculum, BDNF Val66Met for CA4/dentate gyrus, and COMT Val158Met for CA2/3 volume changes. The present study underscores the importance of G × E interactions as determinants of hippocampal volume, which is crucial for the neurobiological understanding of stress-related conditions, such as mood disorders or post-traumatic stress disorder (PTSD).
Assuntos
Interação Gene-Ambiente , Nível de Saúde , Hipocampo/fisiologia , Acontecimentos que Mudam a Vida , Adolescente , Adulto , Feminino , Hipocampo/patologia , Humanos , Masculino , Tamanho do Órgão/fisiologia , Adulto JovemRESUMO
Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a transcriptional coactivator that contributes to the regulation of numerous transcriptional programs including the hepatic response to fasting. Mechanisms at transcriptional and post-transcriptional levels allow PGC-1α to support distinct biological pathways. Here we describe a novel human liver-specific PGC-1α transcript that results from alternative promoter usage and is induced by FOXO1 as well as glucocorticoids and cAMP-response element-binding protein signaling but is not present in other mammals. Hepatic tissue levels of novel and wild-type transcripts were similar but were only moderately associated (p < 0.003). Novel mRNA levels were associated with a polymorphism located in its promoter region, whereas wild-type transcript levels were not. Furthermore, hepatic PCK1 mRNA levels exhibited stronger associations with the novel than with the wild-type transcript levels. Except for a deletion of 127 amino acids at the N terminus, the protein, termed L-PGC-1α, is identical to PGC-1α. L-PGC-1α was localized in the nucleus and showed coactivation properties that overlap with those of PGC-1α. Collectively, our data support a role of L-PGC-1α in gluconeogenesis, but functional differences predicted from the altered structure suggest that L-PGC-1α may have arisen to adapt PGC-1α to more complex metabolic pathways in humans.