RESUMO
Mitochondria are critical for proper organ function and mechanisms to promote mitochondrial health during regeneration would benefit tissue homeostasis. We report that during liver regeneration, proliferation is suppressed in electron transport chain (ETC)-dysfunctional hepatocytes due to an inability to generate acetyl-CoA from peripheral fatty acids through mitochondrial ß-oxidation. Alternative modes for acetyl-CoA production from pyruvate or acetate are suppressed in the setting of ETC dysfunction. This metabolic inflexibility forces a dependence on ETC-functional mitochondria and restoring acetyl-CoA production from pyruvate is sufficient to allow ETC-dysfunctional hepatocytes to proliferate. We propose that metabolic inflexibility within hepatocytes can be advantageous by limiting the expansion of ETC-dysfunctional cells.
Assuntos
Acetilcoenzima A , Hepatócitos , Regeneração Hepática , Mitocôndrias Hepáticas , Ácido Pirúvico , Animais , Hepatócitos/metabolismo , Acetilcoenzima A/metabolismo , Camundongos , Ácido Pirúvico/metabolismo , Mitocôndrias Hepáticas/metabolismo , Oxirredução , Proliferação de Células , Ácidos Graxos/metabolismo , Fígado/metabolismo , Transporte de Elétrons , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , MasculinoRESUMO
Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. Traditionally, it is believed that proliferating cells predominantly rely on de novo synthesis, whereas differentiated tissues favor the salvage pathway. Unexpectedly, we find that adenine and inosine are the most effective circulating precursors for supplying purine nucleotides to tissues and tumors, while hypoxanthine is rapidly catabolized and poorly salvaged in vivo. Quantitative metabolic analysis demonstrates comparative contribution from de novo synthesis and salvage pathways in maintaining purine nucleotide pools in tumors. Notably, feeding mice nucleotides accelerates tumor growth, while inhibiting purine salvage slows down tumor progression, revealing a crucial role of the salvage pathway in tumor metabolism. These findings provide fundamental insights into how normal tissues and tumors maintain purine nucleotides and highlight the significance of purine salvage in cancer.
Assuntos
Neoplasias , Nucleotídeos de Purina , Purinas , Animais , Camundongos , Purinas/metabolismo , Purinas/biossíntese , Neoplasias/metabolismo , Neoplasias/patologia , Nucleotídeos de Purina/metabolismo , Humanos , Inosina/metabolismo , Hipoxantina/metabolismo , Camundongos Endogâmicos C57BL , Adenina/metabolismo , Linhagem Celular Tumoral , FemininoRESUMO
Modulation of the immune response to initiate and halt the inflammatory process occurs both at the site of injury as well as systemically. Due to the evolving role of cellular metabolism in regulating cell fate and function, tendon injuries that undergo normal and aberrant repair were evaluated by metabolic profiling to determine its impact on healing outcomes. Metabolomics revealed an increasing abundance of the immunomodulatory metabolite itaconate within the injury site. Subsequent single-cell RNA-Seq and molecular and metabolomic validation identified a highly mature neutrophil subtype, not macrophages, as the primary producers of itaconate following trauma. These mature itaconate-producing neutrophils were highly inflammatory, producing cytokines that promote local injury fibrosis before cycling back to the bone marrow. In the bone marrow, itaconate was shown to alter hematopoiesis, skewing progenitor cells down myeloid lineages, thereby regulating systemic inflammation. Therapeutically, exogenous itaconate was found to reduce injury-site inflammation, promoting tenogenic differentiation and impairing aberrant vascularization with disease-ameliorating effects. These results present an intriguing role for cycling neutrophils as a sensor of inflammation induced by injury - potentially regulating immune cell production in the bone marrow through delivery of endogenously produced itaconate - and demonstrate a therapeutic potential for exogenous itaconate following tendon injury.
Assuntos
Neutrófilos , Succinatos , Humanos , Neutrófilos/metabolismo , Succinatos/farmacologia , Succinatos/metabolismo , Succinatos/uso terapêutico , Macrófagos/metabolismo , Inflamação/metabolismoRESUMO
The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre Glut3fl/fl) impaired M2 polarization of bone marrow-derived macrophages. Alternatively activated macrophages from the skin of patients with atopic dermatitis showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued in LysM-Cre Glut3fl/fl mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and costaining, and GLUT3 expression was significantly decreased in nonhealing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre Glut3fl/fl mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independently of its glucose transport activity. Unlike plasma membrane-localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL-4Rα subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain, and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport-independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.
Assuntos
Dermatite Atópica , Animais , Humanos , Camundongos , Dermatite Atópica/genética , Endocitose , Glucose/metabolismo , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 3/metabolismo , Interleucina-4/genética , Ativação de Macrófagos/genética , Macrófagos/metabolismo , Cicatrização/genéticaRESUMO
NAD+ kinases (NADKs) are metabolite kinases that phosphorylate NAD+ molecules to make NADP+, a limiting substrate for the generation of reducing power NADPH. NADK2 sustains mitochondrial NADPH production that enables proline biosynthesis and antioxidant defense. However, its molecular architecture and mechanistic regulation remain undescribed. Here, we report the crystal structure of human NADK2, revealing a substrate-driven mode of activation. We find that NADK2 presents an unexpected dimeric organization instead of the typical tetrameric assemblage observed for other NADKs. A specific extended segment (aa 325-365) is crucial for NADK2 dimerization and activity. Moreover, we characterize numerous acetylation events, including those on Lys76 and Lys304, which reside near the active site and inhibit NADK2 activity without disrupting dimerization, thereby reducing mitochondrial NADP(H) production, proline synthesis, and cell growth. These findings reveal important molecular insight into the structure and regulation of a vital enzyme in mitochondrial NADPH and proline metabolism.
Assuntos
Lisina , NAD , Acetilação , Domínio Catalítico , Humanos , Lisina/metabolismo , Proteínas Mitocondriais/metabolismo , NAD/metabolismo , NADP/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Prolina/metabolismoRESUMO
Jouandin et al. (2022) show that lysosomal-derived cysteine serves as a signal to promote the tricarboxylic acid (TCA) cycle and suppress TORC1 signaling for Drosophila to endure starvation periods.
Assuntos
Proteínas de Drosophila , Inanição , Adaptação Psicológica , Animais , Cisteína , Drosophila , Proteínas de Drosophila/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genéticaRESUMO
Purine nucleotides are necessary for various biological processes related to cell proliferation. Despite their importance in DNA and RNA synthesis, cellular signaling, and energy-dependent reactions, the impact of changes in cellular purine levels on cell physiology remains poorly understood. Here, we find that purine depletion stimulates cell migration, despite effective reduction in cell proliferation. Blocking purine synthesis triggers a shunt of glycolytic carbon into the serine synthesis pathway, which is required for the induction of cell migration upon purine depletion. The stimulation of cell migration upon a reduction in intracellular purines required one-carbon metabolism downstream of de novo serine synthesis. Decreased purine abundance and the subsequent increase in serine synthesis triggers an epithelial-mesenchymal transition (EMT) and, in cancer models, promotes metastatic colonization. Thus, reducing the available pool of intracellular purines re-routes metabolic flux from glycolysis into de novo serine synthesis, a metabolic change that stimulates a program of cell migration.
Assuntos
Nucleotídeos de Purina , Serina , Carbono , Movimento Celular , Purinas , Serina/metabolismoRESUMO
The tuberous sclerosis complex (TSC) 1 and 2 proteins associate with TBC1D7 to form the TSC complex, which is an essential suppressor of mTOR complex 1 (mTORC1), a ubiquitous driver of cell and tissue growth. Loss-of-function mutations in TSC1 or TSC2, but not TBC1D7, give rise to TSC, a pleiotropic disorder with aberrant activation of mTORC1 in various tissues. Here, we characterize mice with genetic deletion of Tbc1d7, which are viable with normal growth and development. Consistent with partial loss of function of the TSC complex, Tbc1d7 knockout (KO) mice display variable increases in tissue mTORC1 signaling with increased muscle fiber size but with strength and motor defects. Their most pronounced phenotype is brain overgrowth due to thickening of the cerebral cortex, with enhanced neuron-intrinsic mTORC1 signaling and growth. Thus, TBC1D7 is required for full TSC complex function in tissues, and the brain is particularly sensitive to its growth-suppressing activities.
Assuntos
Encéfalo , Peptídeos e Proteínas de Sinalização Intracelular , Alvo Mecanístico do Complexo 1 de Rapamicina , Neurônios , Proteína 1 do Complexo Esclerose Tuberosa , Esclerose Tuberosa , Proteínas Supressoras de Tumor , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologia , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
OBJECTIVE: The mechanistic target of rapamycin complex 1 (mTORC1) is dynamically regulated by fasting and feeding cycles in the liver to promote protein and lipid synthesis while suppressing autophagy. However, beyond these functions, the metabolic response of the liver to feeding and insulin signaling orchestrated by mTORC1 remains poorly defined. Here, we determine whether ATF4, a stress responsive transcription factor recently found to be independently regulated by mTORC1 signaling in proliferating cells, is responsive to hepatic mTORC1 signaling to alter hepatocyte metabolism. METHODS: ATF4 protein levels and expression of canonical gene targets were analyzed in the liver following fasting and physiological feeding in the presence or absence of the mTORC1 inhibitor, rapamycin. Primary hepatocytes from wild-type or liver-specific Atf4 knockout (LAtf4KO) mice were used to characterize the effects of insulin-stimulated mTORC1-ATF4 function on hepatocyte gene expression and metabolism. Both unbiased steady-state metabolomics and stable-isotope tracing methods were employed to define mTORC1 and ATF4-dependent metabolic changes. RNA-sequencing was used to determine global changes in feeding-induced transcripts in the livers of wild-type versus LAtf4KO mice. RESULTS: We demonstrate that ATF4 and its metabolic gene targets are stimulated by mTORC1 signaling in the liver, in a hepatocyte-intrinsic manner by insulin in response to feeding. While we demonstrate that de novo purine and pyrimidine synthesis is stimulated by insulin through mTORC1 signaling in primary hepatocytes, this regulation was independent of ATF4. Metabolomics and metabolite tracing studies revealed that insulin-mTORC1-ATF4 signaling stimulates pathways of nonessential amino acid synthesis in primary hepatocytes, including those of alanine, aspartate, methionine, and cysteine, but not serine. CONCLUSIONS: The results demonstrate that ATF4 is a novel metabolic effector of mTORC1 in the liver, extending the molecular consequences of feeding and insulin-induced mTORC1 signaling in this key metabolic tissue to the control of amino acid metabolism.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Fígado/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fator 4 Ativador da Transcrição/deficiência , Ração Animal , Animais , Comportamento Alimentar , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transdução de SinaisRESUMO
Nicotinamide adenine dinucleotide phosphate (NADP+) is vital to produce NADPH, a principal supplier of reducing power for biosynthesis of macromolecules and protection against oxidative stress. NADPH exists in separate pools, in both the cytosol and mitochondria; however, the cellular functions of mitochondrial NADPH are incompletely described. Here, we find that decreasing mitochondrial NADP(H) levels through depletion of NAD kinase 2 (NADK2), an enzyme responsible for production of mitochondrial NADP+, renders cells uniquely proline auxotrophic. Cells with NADK2 deletion fail to synthesize proline, due to mitochondrial NADPH deficiency. We uncover the requirement of mitochondrial NADPH and NADK2 activity for the generation of the pyrroline-5-carboxylate metabolite intermediate as the bottleneck step in the proline biosynthesis pathway. Notably, after NADK2 deletion, proline is required to support nucleotide and protein synthesis, making proline essential for the growth and proliferation of NADK2-deficient cells. Thus, we highlight proline auxotrophy in mammalian cells and discover that mitochondrial NADPH is essential to enable proline biosynthesis.
Assuntos
Proliferação de Células , Mitocôndrias/metabolismo , NADP/metabolismo , Prolina/biossíntese , Animais , Ciclo Celular/genética , Humanos , Camundongos , Camundongos Knockout , Consumo de Oxigênio , Pâncreas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The molecular elements that govern cellular transformation and tumorigenic competence remain poorly understood. Metabolic reprogramming has emerged as a hallmark of malignant transformation. Recently in Cell Metabolism, Zhang et al. showed that an increase of cellular antioxidant capacity and nucleotide availability is sufficient to induce oncogenic transformation and tumorigenesis.
Assuntos
Antioxidantes , Nucleotídeos , Carcinogênese , Transformação Celular Neoplásica , HumanosRESUMO
The altered metabolic programme of cancer cells facilitates their cell-autonomous proliferation and survival. In normal cells, signal transduction pathways control core cellular functions, including metabolism, to couple the signals from exogenous growth factors, cytokines or hormones to adaptive changes in cell physiology. The ubiquitous, growth factor-regulated phosphoinositide 3-kinase (PI3K)-AKT signalling network has diverse downstream effects on cellular metabolism, through either direct regulation of nutrient transporters and metabolic enzymes or the control of transcription factors that regulate the expression of key components of metabolic pathways. Aberrant activation of this signalling network is one of the most frequent events in human cancer and serves to disconnect the control of cell growth, survival and metabolism from exogenous growth stimuli. Here we discuss our current understanding of the molecular events controlling cellular metabolism downstream of PI3K and AKT and of how these events couple two major hallmarks of cancer: growth factor independence through oncogenic signalling and metabolic reprogramming to support cell survival and proliferation.
Assuntos
Metabolismo Energético , Neoplasias/etiologia , Neoplasias/metabolismo , Proteínas Oncogênicas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Suscetibilidade a Doenças , Regulação Neoplásica da Expressão Gênica , Glucose/metabolismo , Humanos , Neoplasias/patologia , OxirreduçãoRESUMO
Nicotinamide adenine dinucleotide phosphate (NADP+) is essential for producing NADPH, the primary cofactor for reductive metabolism. We find that growth factor signaling through the phosphoinositide 3-kinase (PI3K)-Akt pathway induces acute synthesis of NADP+ and NADPH. Akt phosphorylates NAD kinase (NADK), the sole cytosolic enzyme that catalyzes the synthesis of NADP+ from NAD+ (the oxidized form of NADH), on three serine residues (Ser44, Ser46, and Ser48) within an amino-terminal domain. This phosphorylation stimulates NADK activity both in cells and directly in vitro, thereby increasing NADP+ production. A rare isoform of NADK (isoform 3) lacking this regulatory region exhibits constitutively increased activity. These data indicate that Akt-mediated phosphorylation of NADK stimulates its activity to increase NADP+ production through relief of an autoinhibitory function inherent to its amino terminus.
Assuntos
NADP/biossíntese , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Animais , Cromatografia Líquida , Citosol/enzimologia , Células HEK293 , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Domínios Proteicos , Serina/genética , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em TandemRESUMO
Mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) integrates signals from growth factors and nutrients to control biosynthetic processes, including protein, lipid, and nucleic acid synthesis. We find that the mTORC1 pathway is responsive to changes in purine nucleotides in a manner analogous to its sensing of amino acids. Depletion of cellular purines, but not pyrimidines, inhibits mTORC1, and restoration of intracellular adenine nucleotides via addition of exogenous purine nucleobases or nucleosides acutely reactivates mTORC1. Adenylate sensing by mTORC1 is dependent on the tuberous sclerosis complex (TSC) protein complex and its regulation of Rheb upstream of mTORC1, but independent of energy stress and AMP-activated protein kinase (AMPK). Even though mTORC1 signaling is not acutely sensitive to changes in intracellular guanylates, long-term depletion of guanylates decreases Rheb protein levels. Our findings suggest that nucleotide sensing, like amino acid sensing, enables mTORC1 to tightly coordinate nutrient availability with the synthesis of macromolecules, such as protein and nucleic acids, produced from those nutrients.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Nucleotídeos de Purina/metabolismo , Células A549 , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos/farmacologia , Fluoruracila/farmacologia , Células HeLa , Humanos , Mercaptopurina/farmacologia , Metotrexato/farmacologia , Camundongos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fosforribosilglicinamido Formiltransferase/antagonistas & inibidores , Fosforribosilglicinamido Formiltransferase/genética , Fosforribosilglicinamido Formiltransferase/metabolismo , Interferência de RNA , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
This corrects the article DOI: 10.1038/nature20789.
RESUMO
Metformin is the most widely prescribed drug for the treatment of type 2 diabetes. However, knowledge of the full effects of metformin on biochemical pathways and processes in its primary target tissue, the liver, is limited. One established effect of metformin is to decrease cellular energy levels. The AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) are key regulators of metabolism that are respectively activated and inhibited in acute response to cellular energy depletion. Here we show that metformin robustly inhibits mTORC1 in mouse liver tissue and primary hepatocytes. Using mouse genetics, we find that at the lowest concentrations of metformin that inhibit hepatic mTORC1 signaling, this inhibition is dependent on AMPK and the tuberous sclerosis complex (TSC) protein complex (TSC complex). Finally, we show that metformin profoundly inhibits hepatocyte protein synthesis in a manner that is largely dependent on its ability to suppress mTORC1 signaling.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fígado/metabolismo , Metformina/farmacologia , Complexos Multiproteicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Esclerose Tuberosa/metabolismo , Animais , Relação Dose-Resposta a Droga , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
Ageing is driven by a loss of transcriptional and protein homeostasis and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction associated with age therefore have the potential to reduce overall disease risk in the elderly. Precursor mRNA (pre-mRNA) splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to several age-related chronic illnesses. However, the role of splicing homeostasis in healthy ageing remains unclear. Here we demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, we find defects in global pre-mRNA splicing with age that are reduced by dietary restriction via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP). We show that SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. We also demonstrate that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in dietary restriction longevity and suggest that modulation of specific spliceosome components may prolong healthy ageing.