RESUMO
Metabolic reprogramming is one of the main hallmarks of cancer cells. It refers to the metabolic adaptations of tumor cells in response to nutrient deficiency, microenvironmental insults, and anti-cancer therapies. Metabolic transformation during tumor development plays a critical role in the continued tumor growth and progression and is driven by a complex interplay between the tumor mutational landscape, epigenetic modifications, and microenvironmental influences. Understanding the tumor metabolic vulnerabilities might open novel diagnostic and therapeutic approaches with the potential to improve the efficacy of current tumor treatments. Prostate cancer is a highly heterogeneous disease harboring different mutations and tumor cell phenotypes. While the increase of intra-tumor genetic and epigenetic heterogeneity is associated with tumor progression, less is known about metabolic regulation of prostate cancer cell heterogeneity and plasticity. This review summarizes the central metabolic adaptations in prostate tumors, state-of-the-art technologies for metabolic analysis, and the perspectives for metabolic targeting and diagnostic implications.
Assuntos
Neoplasias da Próstata , Epigênese Genética , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismoRESUMO
Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are important to these processes. Although much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism, little is understood about the role of fibroblast growth factors (FGFs) in this context. Here we identify FGF receptor (FGFR) signalling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signalling inputs results in decreased glycolysis, leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/Fgfr3 double mutant mice, while HK2 overexpression partly rescues the defects caused by suppression of FGF signalling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Glicólise , Neovascularização Fisiológica , Transdução de Sinais , Animais , Movimento Celular , Proliferação de Células , Feminino , Hexoquinase/metabolismo , Linfangiogênese , Vasos Linfáticos/citologia , Vasos Linfáticos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
Histamine-induced vascular leakage is a core process of allergic pathologies, including anaphylaxis. Here, we show that glycolysis is integral to histamine-induced endothelial barrier disruption and hyperpermeability. Histamine rapidly enhanced glycolysis in endothelial cells via a pathway that involved histamine receptor 1 and phospholipase C beta signaling. Consistently, partial inhibition of glycolysis with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) prevented histamine-induced hyperpermeability in human microvascular endothelial cells, by abolishing the histamine-induced actomyosin contraction, focal adherens junction formation, and endothelial barrier disruption. Pharmacologic blockade of glycolysis with 3PO in mice reduced histamine-induced vascular hyperpermeability, prevented vascular leakage in passive cutaneous anaphylaxis and protected from systemic anaphylaxis. In conclusion, we elucidated the role of glycolysis in histamine-induced disruption of endothelial barrier integrity. Our data thereby point to endothelial glycolysis as a novel therapeutic target for human pathologies related to excessive vascular leakage, such as systemic anaphylaxis.
Assuntos
Permeabilidade Capilar/fisiologia , Células Endoteliais/efeitos dos fármacos , Glicólise/fisiologia , Histamina/farmacologia , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Anafilaxia/metabolismo , Anafilaxia/patologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Camundongos , Fosfolipase C beta/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Previous studies have implicated age-associated reductions in mitochondrial oxidative phosphorylation activity in skeletal muscle as a predisposing factor for intramyocellular lipid (IMCL) accumulation and muscle insulin resistance (IR) in the elderly. To further investigate potential alterations in muscle mitochondrial function associated with aging, we assessed basal and insulin-stimulated rates of muscle pyruvate dehydrogenase (VPDH) flux relative to citrate synthase flux (VCS) in healthy lean, elderly subjects and healthy young body mass index- and activity-matched subjects. VPDH/VCS flux was assessed from the (13)C incorporation from of infused [1-13C] glucose into glutamate [4-13C] relative to alanine [3-13C] assessed by LC-tandem MS in muscle biopsies. Insulin-stimulated rates of muscle glucose uptake were reduced by 25% (P<0.01) in the elderly subjects and were associated with â¼70% (P<0.04) increase in IMCL, assessed by 1H magnetic resonance spectroscopy. Basal VPDH/VCS fluxes were similar between the groups (young: 0.20±0.03; elderly: 0.14±0.03) and increased approximately threefold in the young subjects following insulin stimulation. However, this increase was severely blunted in the elderly subjects (young: 0.55±0.04; elderly: 0.18±0.02, P=0.0002) and was associated with an â¼40% (P=0.004) reduction in insulin activation of Akt. These results provide new insights into acquired mitochondrial abnormalities associated with aging and demonstrate that age-associated reductions in muscle mitochondrial function and increased IMCL are associated with a marked inability of mitochondria to switch from lipid to glucose oxidation during insulin stimulation.
Assuntos
Envelhecimento , Glucose/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Adulto , Idoso , Glicemia/metabolismo , Isótopos de Carbono , Cromatografia Líquida , Citrato (si)-Sintase/metabolismo , Técnica Clamp de Glucose , Humanos , Hipoglicemiantes/farmacologia , Insulina/sangue , Insulina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Mitocôndrias/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Complexo Piruvato Desidrogenase/metabolismo , Espectrometria de Massas em TandemRESUMO
In mammals, pyruvate kinase (PK) plays a key role in regulating the balance between glycolysis and gluconeogenesis; however, in vivo regulation of PK flux by gluconeogenic hormones and substrates is poorly understood. To this end, we developed a novel NMR-liquid chromatography/tandem-mass spectrometry (LC-MS/MS) method to directly assess pyruvate cycling relative to mitochondrial pyruvate metabolism (VPyr-Cyc/VMito) in vivo using [3-(13)C]lactate as a tracer. Using this approach, VPyr-Cyc/VMito was only 6% in overnight fasted rats. In contrast, when propionate was infused simultaneously at doses previously used as a tracer, it increased VPyr-Cyc/VMito by 20-30-fold, increased hepatic TCA metabolite concentrations 2-3-fold, and increased endogenous glucose production rates by 20-100%. The physiologic stimuli, glucagon and epinephrine, both increased hepatic glucose production, but only glucagon suppressed VPyr-Cyc/VMito These data show that under fasting conditions, when hepatic gluconeogenesis is stimulated, pyruvate recycling is relatively low in liver compared with VMito flux and that liver metabolism, in particular pyruvate cycling, is sensitive to propionate making it an unsuitable tracer to assess hepatic glycolytic, gluconeogenic, and mitochondrial metabolism in vivo.
Assuntos
Ciclo do Ácido Cítrico/efeitos dos fármacos , Fígado/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Propionatos/farmacologia , Ácido Pirúvico/metabolismo , Animais , Glicemia/metabolismo , Cromatografia Líquida , Epinefrina/sangue , Epinefrina/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Glucagon/sangue , Glucagon/farmacologia , Gluconeogênese/efeitos dos fármacos , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Insulina/sangue , Fígado/metabolismo , Mitocôndrias/metabolismo , Propionatos/administração & dosagem , Ratos Sprague-Dawley , Espectrometria de Massas em TandemRESUMO
Hepatic insulin resistance is a principal component of type 2 diabetes, but the cellular and molecular mechanisms responsible for its pathogenesis remain unknown. Recent studies have suggested that saturated fatty acids induce hepatic insulin resistance through activation of the toll-like receptor 4 (TLR-4) receptor in the liver, which in turn transcriptionally activates hepatic ceramide synthesis leading to inhibition of insulin signaling. In this study, we demonstrate that TLR-4 receptor signaling is not directly required for saturated or unsaturated fat-induced hepatic insulin resistance in both TLR-4 antisense oligonucleotide treated and TLR-4 knockout mice, and that ceramide accumulation is not dependent on TLR-4 signaling or a primary event in hepatic steatosis and impairment of insulin signaling. Further, we show that both saturated and unsaturated fats lead to hepatic accumulation of diacylglycerols, activation of PKCε, and impairment of insulin-stimulated IRS-2 signaling. These data demonstrate that saturated fat-induced insulin resistance is independent of TLR-4 activation and ceramides.
Assuntos
Gorduras Insaturadas na Dieta/farmacologia , Fígado Gorduroso/metabolismo , Resistência à Insulina , Fígado/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Diglicerídeos/metabolismo , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/patologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Fígado/patologia , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
Synthesis of phosphoenolpyruvate (PEP) from oxaloacetate is an absolute requirement for gluconeogenesis from mitochondrial substrates. Generally, this reaction has solely been attributed to the cytosolic isoform of PEPCK (PEPCK-C), although loss of the mitochondrial isoform (PEPCK-M) has never been assessed. Despite catalyzing the same reaction, to date the only significant role reported in mammals for the mitochondrial isoform is as a glucose sensor necessary for insulin secretion. We hypothesized that this nutrient-sensing mitochondrial GTP-dependent pathway contributes importantly to gluconeogenesis. PEPCK-M was acutely silenced in gluconeogenic tissues of rats using antisense oligonucleotides both in vivo and in isolated hepatocytes. Silencing PEPCK-M lowers plasma glucose, insulin, and triglycerides, reduces white adipose, and depletes hepatic glycogen, but raises lactate. There is a switch of gluconeogenic substrate preference to glycerol that quantitatively accounts for a third of glucose production. In contrast to the severe mitochondrial deficiency characteristic of PEPCK-C knock-out livers, hepatocytes from PEPCK-M-deficient livers maintained normal oxidative function. Consistent with its predicted role, gluconeogenesis rates from hepatocytes lacking PEPCK-M are severely reduced for lactate, alanine, and glutamine, but not for pyruvate and glycerol. Thus, PEPCK-M has a direct role in fasted and fed glucose homeostasis, and this mitochondrial GTP-dependent pathway should be reconsidered for its involvement in both normal and diabetic metabolism.
Assuntos
Regulação Enzimológica da Expressão Gênica , Gluconeogênese , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fígado/enzimologia , Fígado/metabolismo , Mitocôndrias/enzimologia , Fosfoenolpiruvato Carboxiquinase (GTP)/fisiologia , Ração Animal , Animais , Glicemia/metabolismo , Privação de Alimentos , Inativação Gênica , Glicerol/metabolismo , Glicogênio/metabolismo , Guanosina Trifosfato/metabolismo , Hepatócitos/citologia , Homeostase , Insulina/metabolismo , Isoenzimas/fisiologia , Ácido Láctico/metabolismo , Masculino , Mitocôndrias/metabolismo , Oligonucleotídeos Antissenso/química , Oxigênio/metabolismo , Consumo de Oxigênio , Ratos , Ratos Sprague-DawleyRESUMO
Fatty acid amide hydrolase (FAAH) knockout mice are prone to excess energy storage and adiposity, whereas mutations in FAAH are associated with obesity in humans. However, the molecular mechanism by which FAAH affects energy expenditure (EE) remains unknown. Here we show that reduced energy expenditure in FAAH(-/-) mice could be attributed to decreased circulating triiodothyronine and thyroxine concentrations secondary to reduced mRNA expression of both pituitary thyroid-stimulating hormone and hypothalamic thyrotropin-releasing hormone. These reductions in the hypothalamic-pituitary-thyroid axis were associated with activation of hypothalamic peroxisome proliferating-activated receptor γ (PPARγ), and increased hypothalamic deiodinase 2 expression. Infusion of NAEs (anandamide and palmitoylethanolamide) recapitulated increases in PPARγ-mediated decreases in EE. FAAH(-/-) mice were also prone to diet-induced hepatic insulin resistance, which could be attributed to increased hepatic diacylglycerol content and protein kinase Cε activation. Our data indicate that FAAH deletion, and the resulting increases in NAEs, predispose mice to ectopic lipid storage and hepatic insulin resistance by promoting centrally mediated hypothyroidism.
Assuntos
Amidoidrolases/genética , Metabolismo Energético/fisiologia , Hipotireoidismo/complicações , Hipotireoidismo/genética , Resistência à Insulina/fisiologia , Amidas , Amidoidrolases/deficiência , Análise de Variância , Animais , Ácidos Araquidônicos/administração & dosagem , Cromatografia Líquida , Endocanabinoides/administração & dosagem , Metabolismo Energético/genética , Etanolaminas/administração & dosagem , Hipotireoidismo/enzimologia , Immunoblotting , Camundongos , Camundongos Knockout , PPAR gama , Ácidos Palmíticos/administração & dosagem , Reação em Cadeia da Polimerase , Alcamidas Poli-Insaturadas/administração & dosagem , Espectrometria de Massas em Tandem , Tireotropina/metabolismo , Hormônio Liberador de Tireotropina/metabolismo , Tiroxina/sangue , Tri-Iodotironina/sangueRESUMO
Uncoupling protein-3 (UCP3) is a mitochondrial transmembrane protein highly expressed in the muscle that has been implicated in regulating the efficiency of mitochondrial oxidative phosphorylation. Increasing UCP3 expression in skeletal muscle enhances proton leak across the inner mitochondrial membrane and increases oxygen consumption in isolated mitochondria, but its precise function in vivo has yet to be fully elucidated. To examine whether muscle-specific overexpression of UCP3 modulates muscle mitochondrial oxidation in vivo, rates of ATP synthesis were assessed by 31 P magnetic resonance spectroscopy (MRS), and rates of mitochondrial oxidative metabolism were measured by assessing the rate of [2-13 C]acetate incorporation into muscle [4-13 C]-, [3-13 C]-glutamate, and [4-13 C]-glutamine by high-resolution 13 C/1 H MRS. Using this approach, we found that the overexpression of UCP3 in skeletal muscle was accompanied by increased muscle mitochondrial inefficiency in vivo as reflected by a 42% reduction in the ratio of ATP synthesis to mitochondrial oxidation.
Assuntos
Canais Iônicos , Mitocôndrias , Animais , Camundongos , Trifosfato de Adenosina/metabolismo , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias Musculares , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Prótons , Proteína Desacopladora 3/análise , Proteína Desacopladora 3/metabolismoRESUMO
OBJECTIVE: Rheumatoid arthritis is an inflammatory joint disease in which synovial iron deposition has been described. Transferrin receptor 2 (Tfr2) represents a critical regulator of systemic iron levels. Loss of Tfr2 function in humans and mice results in iron overload. As iron contributes to inflammatory processes, we investigated whether Tfr2-deletion affects the pathogenesis of inflammatory arthritis in an iron-dependent manner. METHODS: Using a global and conditional genetic disruption of Tfr2, we assessed the relevance of Tfr2 in K/BxN serum-transfer arthritis (STA) and macrophage polarization. RESULTS: Male Tfr2-/- mice subjected to STA developed pronounced joint swelling, and bone erosion as compared to Tfr2+/+ littermate-controls (P < 0.01). Furthermore, an increase of neutrophils and macrophages/monocytes was observed in the inflammatory infiltrate within the paws of Tfr2-/- mice. To elucidate whether Tfr2 in myeloid cells has a direct role in the pathogenesis of arthritis or whether the effects were mediated via the systemic iron overload, we induced STA in Tfr2fl/fl-LysMCre + mice, which showed normal iron-loading. Cre + female mice displayed increased disease development compared to Cre-controls. As macrophages regulate iron availability and innate immunity, we hypothesized that Tfr2-deficiency would polarize macrophages toward a pro-inflammatory state (M1) that contributes to arthritis progression. In response to IFN-γ stimulation, Tfr2-/- macrophages showed increased expression of M1-like cytokines, IFN-γ-target genes, nitric-oxide production, and prolonged STAT1 activation compared to Tfr2+/+ macrophages (P < 0.01), while pre-treatment with ruxolitinib abolished Tfr2-driven M1-like polarization. CONCLUSION: Taken together, these findings suggest a protective role of Tfr2 in macrophages on the progression of arthritis via suppression of M1-like polarization.
Assuntos
Artrite , Sobrecarga de Ferro , Humanos , Camundongos , Masculino , Feminino , Animais , Camundongos Knockout , Ferro/metabolismo , Sobrecarga de Ferro/patologia , Macrófagos/metabolismo , Artrite/metabolismo , Receptores da Transferrina/genéticaRESUMO
BACKGROUND: The detrimental impact of malnutrition and cachexia in cancer patients subjected to surgical resection is well established. However, how systemic and local metabolic alterations in cancer patients impact the serum metabolite signature, thereby leading to cancer-specific differences, is poorly defined. In order to implement metabolomics as a potential tool in clinical diagnostics and disease follow-up, targeted metabolite profiling based on quantitative measurements is essential. We hypothesized that the quantitative metabolic profile assessed by 1 H nuclear magnetic resonance (NMR) spectroscopy can be used to identify cancer-induced catabolism and potentially distinguish between specific tumour entities. Importantly, to prove tumour dependency and assess metabolic normalization, we additionally analysed the metabolome of patients' sera longitudinally post-surgery in order to assess metabolic normalization. METHODS: Forty two metabolites in sera of patients with tumour entities known to cause malnutrition and cachexia, namely, upper gastrointestinal cancer and pancreatic cancer, as well as sera of healthy controls, were quantified by 1 H NMR spectroscopy. RESULTS: Comparing serum metabolites of patients with gastrointestinal cancer with healthy controls and pancreatic cancer patients, we identified at least 15 significantly changed metabolites in each comparison. Principal component and pathway analysis tools showed a catabolic signature in preoperative upper gastrointestinal cancer patients. The most specifically upregulated metabolite group in gastrointestinal cancer patients was ketone bodies (3-hydroxybutyrate, P < 0.0001; acetoacetate, P < 0.0001; acetone, P < 0.0001; false discovery rate [FDR] adjusted). Increased glycerol levels (P < 0.0001), increased concentration of the ketogenic amino acid lysine (P = 0.03) and a significant correlation of 3-hydroxybutyrate levels with branched-chained amino acids (leucine, P = 0.02; isoleucine, P = 0.04 [FDR adjusted]) suggested that ketone body synthesis was driven by lipolysis and amino acid breakdown. Interestingly, the catabolic signature was independent of the body mass index, clinically assessed malnutrition using the nutritional risk screening score, and systemic inflammation assessed by CRP and leukocyte count. Longitudinal measurements and principal component analyses revealed a quick normalization of key metabolic alterations seven days post-surgery, including ketosis. CONCLUSIONS: Together, the quantitative metabolic profile obtained by 1 H NMR spectroscopy identified a tumour-induced catabolic signature specific to upper gastrointestinal cancer patients and enabled monitoring restoration of metabolic homeostasis after surgery. This approach was critical to identify the obtained metabolic profile as an upper gastrointestinal cancer-specific signature independent of malnutrition and inflammation.
Assuntos
Neoplasias Gastrointestinais , Desnutrição , Neoplasias Pancreáticas , Humanos , Ácido 3-Hidroxibutírico , Caquexia/etiologia , Caquexia/metabolismo , Neoplasias Gastrointestinais/complicações , Neoplasias Gastrointestinais/metabolismo , Inflamação/metabolismo , Leucina , Desnutrição/etiologia , Desnutrição/metabolismo , Neoplasias Pancreáticas/metabolismo , MetabolômicaRESUMO
UNLABELLED: Pyruvate dehydrogenase plays a critical role in the regulation of hepatic glucose and fatty acid oxidation; however, surprisingly little is known about its regulation in vivo. In this study we examined the individual effects of insulin and substrate availability on the regulation of pyruvate dehydrogenase flux (V(PDH) ) to tricarboxylic acid flux (V(TCA) ) in livers of awake rats with lipid-induced hepatic insulin resistance. V(PDH) /V(TCA) flux was estimated from the [4-(13) C]glutamate/[3-(13) C]alanine enrichments in liver extracts and assessed under conditions of fasting and during a hyperinsulinemic-euglycemic clamp, whereas the effects of increased plasma glucose concentration on V(PDH) /V(TCA) flux was assessed during a hyperglycemic clamp in conjunction with infusions of somatostatin and insulin to maintain basal concentrations of insulin. The effects of increases in both glucose and insulin on V(PDH) /V(TCA) were examined during a hyperinsulinemic-hyperglycemic clamp. The effects of chronic lipid-induced hepatic insulin resistance on this flux were also examined by performing these measurements in rats fed a high-fat diet for 3 weeks. Using this approach we found that fasting V(PDH) /V(TCA) was reduced by 95% in rats with hepatic insulin resistance (from 17.2 ± 1.5% to 1.3 ± 0.7%, P < 0.00001). Surprisingly, neither hyperinsulinemia per se or hyperglycemia per se were sufficient to increase V(PDH) /V(TCA) flux. Only under conditions of combined hyperglycemia and hyperinsulinemia did V(PDH) /V(TCA) flux increase (44.6 ± 3.2%, P < 0.0001 versus basal) in low-fat fed animals but not in rats with chronic lipid-induced hepatic insulin resistance. CONCLUSION: These studies demonstrate that the combination of both hyperinsulinemia and hyperglycemia are required to increase V(PDH) /V(TCA) flux in vivo and that this flux is severely diminished in rats with chronic lipid-induced hepatic insulin resistance.
Assuntos
Gorduras/metabolismo , Glucose/metabolismo , Resistência à Insulina/fisiologia , Fígado/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Animais , Glicemia/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Gorduras na Dieta/administração & dosagem , Técnica Clamp de Glucose , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
In the presence of high abundance of exogenous fatty acids, cells either store fatty acids in lipid droplets or oxidize them in mitochondria. In this study, we aimed to explore a novel and direct role of mitochondrial fission in lipid homeostasis in HeLa cells. We observed the association between mitochondrial morphology and lipid droplet accumulation in response to high exogenous fatty acids. We inhibited mitochondrial fission by silencing dynamin-related protein 1(DRP1) and observed the shift in fatty acid storage-usage balance. Inhibition of mitochondrial fission resulted in an increase in fatty acid content of lipid droplets and a decrease in mitochondrial fatty acid oxidation. Next, we overexpressed carnitine palmitoyltransferase-1 (CPT1), a key mitochondrial protein in fatty acid oxidation, to further examine the relationship between mitochondrial fatty acid usage and mitochondrial morphology. Mitochondrial fission plays a role in distributing exogenous fatty acids. CPT1A controlled the respiratory rate of mitochondrial fatty acid oxidation but did not cause a shift in the distribution of fatty acids between mitochondria and lipid droplets. Our data reveals a novel function for mitochondrial fission in balancing exogenous fatty acids between usage and storage, assigning a role for mitochondrial dynamics in control of intracellular fuel utilization and partitioning.
RESUMO
Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Specific metabolic features of PCa might serve as therapeutic targets for tumor radiosensitization and as biomarkers for identifying the patients most likely to respond to radiotherapy. The study aimed to characterize a potential role of glutaminase (GLS)-driven glutamine catabolism as a prognostic biomarker and a therapeutic target for PCa radiosensitization. Methods: We analyzed primary cell cultures and radioresistant (RR) derivatives of the conventional PCa cell lines by gene expression and metabolic assays to identify the molecular traits associated with radiation resistance. Relative radiosensitivity of the cell lines and primary cell cultures were analyzed by 2-D and 3-D clonogenic analyses. Targeting of glutamine (Gln) metabolism was achieved by Gln starvation, gene knockdown, and chemical inhibition. Activation of the DNA damage response (DDR) and autophagy was assessed by gene expression, western blotting, and fluorescence microscopy. Reactive oxygen species (ROS) and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were analyzed by fluorescence and luminescence probes, respectively. Cancer stem cell (CSC) properties were investigated by sphere-forming assay, CSC marker analysis, and in vivo limiting dilution assays. Single circulating tumor cells (CTCs) isolated from the blood of PCa patients were analyzed by array comparative genome hybridization. Expression levels of the GLS1 and MYC gene in tumor tissues and amino acid concentrations in blood plasma were correlated to a progression-free survival in PCa patients. Results: Here, we found that radioresistant PCa cells and prostate CSCs have a high glutamine demand. GLS-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as GLS and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy. Conclusions: Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.
Assuntos
Glutamina/metabolismo , Neoplasias da Próstata/metabolismo , Tolerância a Radiação/genética , Animais , Autofagia , Proteína 5 Relacionada à Autofagia/metabolismo , Biomarcadores Farmacológicos , Linhagem Celular Tumoral , Glutaminase/antagonistas & inibidores , Glutaminase/genética , Glutaminase/metabolismo , Humanos , Masculino , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Oxirredução , Proteínas Proto-Oncogênicas c-myc/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Stem cell-derived ß (SC-ß) cells could provide unlimited human ß cells toward a curative diabetes treatment. Differentiation of SC-ß cells yields transplantable islets that secrete insulin in response to glucose challenges. Following transplantation into mice, SC-ß cell function is comparable to human islets, but the magnitude and consistency of response in vitro are less robust than observed in cadaveric islets. Here, we profile metabolism of SC-ß cells and islets to quantify their capacity to sense glucose and identify reduced anaplerotic cycling in the mitochondria as the cause of reduced glucose-stimulated insulin secretion in SC-ß cells. This activity can be rescued by challenging SC-ß cells with intermediate metabolites from the TCA cycle and late but not early glycolysis, downstream of the enzymes glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase. Bypassing this metabolic bottleneck results in a robust, bi-phasic insulin release in vitro that is identical in magnitude to functionally mature human islets.
Assuntos
Linfócitos B/metabolismo , Glucose/metabolismo , Glicólise/genética , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Humanos , CamundongosRESUMO
The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle couples mitochondrial PEPCK (PCK2) to pyruvate kinase (PK) in the liver and pancreatic islets to regulate glucose homeostasis. Here, small molecule PK activators accelerated the PEP cycle to improve islet function, as well as metabolic homeostasis, in preclinical rodent models of diabetes. In contrast, treatment with a PK activator did not improve insulin secretion in pck2-/- mice. Unlike other clinical secretagogues, PK activation enhanced insulin secretion but also had higher insulin content and markers of differentiation. In addition to improving insulin secretion, acute PK activation short-circuited gluconeogenesis to reduce endogenous glucose production while accelerating red blood cell glucose turnover. Four-week delivery of a PK activator in vivo remodeled PK phosphorylation, reduced liver fat, and improved hepatic and peripheral insulin sensitivity in HFD-fed rats. These data provide a preclinical rationale for PK activation to accelerate the PEP cycle to improve metabolic homeostasis and insulin sensitivity.
Assuntos
Mitocôndrias/metabolismo , Fosfoenolpiruvato/metabolismo , Animais , Homeostase , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Piruvato Quinase/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Alterations in muscle mitochondrial substrate preference have been postulated to play a major role in the pathogenesis of muscle insulin resistance. In order to examine this hypothesis, we assessed the ratio of mitochondrial pyruvate oxidation (VPDH) to rates of mitochondrial citrate synthase flux (VCS) in muscle. Contrary to this hypothesis, we found that high-fat-diet (HFD)-fed insulin-resistant rats did not manifest altered muscle substrate preference (VPDH/VCS) in soleus or quadriceps muscles in the fasting state. Furthermore, hyperinsulinemic-euglycemic (HE) clamps increased VPDH/VCS in both muscles in normal and insulin-resistant rats. We then examined the muscle VPDH/VCS flux in insulin-sensitive and insulin-resistant humans and found similar relative rates of VPDH/VCS, following an overnight fast (â¼20%), and similar increases in VPDH/VCS fluxes during a HE clamp. Altogether, these findings demonstrate that alterations in mitochondrial substrate preference are not an essential step in the pathogenesis of muscle insulin resistance.
Assuntos
Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Adulto , Animais , Humanos , Resistência à Insulina , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Pancreatic ß cells couple nutrient metabolism with appropriate insulin secretion. Here, we show that pyruvate kinase (PK), which converts ADP and phosphoenolpyruvate (PEP) into ATP and pyruvate, underlies ß cell sensing of both glycolytic and mitochondrial fuels. Plasma membrane-localized PK is sufficient to close KATP channels and initiate calcium influx. Small-molecule PK activators increase the frequency of ATP/ADP and calcium oscillations and potently amplify insulin secretion. PK restricts respiration by cyclically depriving mitochondria of ADP, which accelerates PEP cycling until membrane depolarization restores ADP and oxidative phosphorylation. Our findings support a compartmentalized model of ß cell metabolism in which PK locally generates the ATP/ADP required for insulin secretion. Oscillatory PK activity allows mitochondria to perform synthetic and oxidative functions without any net impact on glucose oxidation. These findings suggest a potential therapeutic route for diabetes based on PK activation that would not be predicted by the current consensus single-state model of ß cell function.
Assuntos
Insulina/metabolismo , Piruvato Quinase/metabolismo , Animais , Linhagem Celular , Humanos , Secreção de Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Mechanisms coordinating pancreatic ß cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of ß cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phosphoenolpyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and ß cell health.
Assuntos
Trifosfato de Adenosina/metabolismo , Glucose/metabolismo , Guanosina Trifosfato/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocôndrias/metabolismo , Succinato-CoA Ligases/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Ciclo do Ácido Cítrico/genética , Homeostase , Humanos , Secreção de Insulina/genética , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Fosforilação Oxidativa , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Regulação para CimaRESUMO
Metabolism is pivotal for formation of the lymphatic vasculature. Understanding metabolism in lymphatic endothelial cells (LECs) requires quantitative characterization of specific metabolic pathways. Here we describe methods for using radioactive tracers to assess flux rates of glycolysis, fatty acid ß-oxidation, glucose oxidation, and glutamine oxidation. We also provide a detailed method for utilizing mass spectrometry (MS) to measure glycolytic intermediates and ATP.