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1.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073144

RESUMO

Angiogenesis is the process of new blood vessel formation. In this complex orchestrated growth, many factors are included. Lately, focus has shifted to endothelial cell metabolism, particularly to the PFKFB3 protein, a key regulatory enzyme of the glycolytic pathway. A variety of inhibitors of this important target have been studied, and a plethora of biological effects related to the process of angiogenesis have been reported. However, recent studies have disputed their mechanism of action, questioning whether all the effects are indeed due to PFKFB3 inhibition. Remarkably, the most well-studied inhibitor, 3PO, does not bind to PFKFB3, raising questions about this target. In our study, we aimed to elucidate the effects of PFKFB3 inhibition in angiogenesis by using the small molecule AZ67. We used isothermal titration calorimetry and confirmed binding to PFKFB3. In vitro, AZ67 did not decrease lactate production in endothelial cells (ECs), nor ATP levels, but exhibited good inhibitory efficacy in the tube-formation assay. Surprisingly, this was independent of EC migratory and proliferative abilities, as this was not diminished upon treatment. Strikingly however, even the lowest dose of AZ67 demonstrated significant inhibition of angiogenesis in vivo. To our knowledge, this is the first study to demonstrate that the process of angiogenesis can be disrupted by targeting PFKFB3 independently of glycolysis inhibition.


Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos , Glicólise/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Fosfofrutoquinase-2 , Animais , Linhagem Celular , Células Endoteliais , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosfofrutoquinase-2/antagonistas & inibidores , Fosfofrutoquinase-2/metabolismo , Ligação Proteica
2.
FASEB J ; 35(7): e21728, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34110658

RESUMO

Proliferation and differentiation of preadipocytes, and other cell types, is accompanied by an increase in glucose uptake. Previous work showed that a pulse of high glucose was required during the first 3 days of differentiation in vitro, but was not required after that. The specific glucose metabolism pathways required for adipocyte differentiation are unknown. Herein, we used 3T3-L1 adipocytes as a model system to study glucose metabolism and expansion of the adipocyte metabolome during the first 3 days of differentiation. Our primary outcome measures were GLUT4 and adiponectin, key proteins associated with healthy adipocytes. Using complete media with 0 or 5 mM glucose, we distinguished between developmental features that were dependent on the differentiation cocktail of dexamethasone, insulin, and isobutylmethylxanthine alone or the cocktail plus glucose. Cocktail alone was sufficient to activate the capacity for 2-deoxglucose uptake and glycolysis, but was unable to support the expression of GLUT4 and adiponectin in mature adipocytes. In contrast, 5 mM glucose in the media promoted a transient increase in glucose uptake and glycolysis as well as a significant expansion of the adipocyte metabolome and proteome. Using genetic and pharmacologic approaches, we found that the positive effects of 5 mM glucose on adipocyte differentiation were specifically due to increased expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of glycolysis and the ancillary glucose metabolic pathways. Our data reveal a critical role for PFKFB3 activity in regulating the cellular metabolic remodeling required for adipocyte differentiation and maturation.


Assuntos
Adipócitos/metabolismo , Glucose/metabolismo , Fosfofrutoquinase-2/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adiponectina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Dexametasona/farmacologia , Transportador de Glucose Tipo 4/metabolismo , Glicólise/efeitos dos fármacos , Glicólise/fisiologia , Insulina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Xantinas/farmacologia
3.
J Nutr Biochem ; 95: 108764, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33964465

RESUMO

Obesity-associated inflammation in white adipose tissue (WAT) is a causal factor of systemic insulin resistance. To better understand how adipocytes regulate WAT inflammation, the present study generated chimeric mice in which inducible 6-phosphofructo-2-kinase was low, normal, or high in WAT while the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (Pfkfb3) was normal in hematopoietic cells, and analyzed changes in high-fat diet (HFD)-induced WAT inflammation and systemic insulin resistance in the mice. Indicated by proinflammatory signaling and cytokine expression, the severity of HFD-induced WAT inflammation in WT â†’ Pfkfb3+/- mice, whose Pfkfb3 was disrupted in WAT adipocytes but not hematopoietic cells, was comparable with that in WT â†’ WT mice, whose Pfkfb3 was normal in all cells. In contrast, the severity of HFD-induced WAT inflammation in WT â†’ Adi-Tg mice, whose Pfkfb3 was over-expressed in WAT adipocytes but not hematopoietic cells, remained much lower than that in WT â†’ WT mice. Additionally, HFD-induced insulin resistance was correlated with the status of WAT inflammation and comparable between WT â†’ Pfkfb3+/- mice and WT â†’ WT mice, but was significantly lower in WT â†’ Adi-Tg mice than in WT â†’ WT mice. In vitro, palmitoleate decreased macrophage phosphorylation states of Jnk p46 and Nfkb p65 and potentiated the effect of interleukin 4 on suppressing macrophage proinflammatory activation. Taken together, these results suggest that the Pfkfb3 in adipocytes functions to suppress WAT inflammation. Moreover, the role played by adipocyte Pfkfb3 is attributable to, at least in part, palmitoleate promotion of macrophage anti-inflammatory activation.


Assuntos
Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Inflamação/metabolismo , Macrófagos/fisiologia , Fosfofrutoquinase-2/metabolismo , Adipócitos Brancos/metabolismo , Transferência Adotiva , Animais , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/farmacologia , Ácidos Graxos Monoinsaturados/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Intolerância à Glucose , Resistência à Insulina , Masculino , Camundongos , Fosfofrutoquinase-2/genética , Transdução de Sinais
4.
Aging (Albany NY) ; 13(10): 14355-14371, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016793

RESUMO

In the present study, the effects and mechanism of action of U50,488H (a selective κ-opioid receptor agonist) on calcification of rat vascular smooth muscle cells (VSMCs) induced by ß-glycerophosphate (ß-GP) were investigated. VSMCs were isolated and cultured in traditional FBS-based media. A calcification model was established in VSMCs under hyperphosphatemia and intracellular calcium contents. Alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and lactate were detected in cell culture supernatants before and after treatment. Alizarin red staining was used to detect the degree of calcification of VSMCs. Expression levels of key molecules of osteogenic markers, fructose-2,6-biphosphatase 3 (PFKFB3), and proline hydroxylase 2 (PHD2), were determined using western blotting. Further, vascular calcification was induced by vitamin D3 plus nicotine in rats and isolated thoracic aortas, calcium concentration was assessed in rat aortic rings in vitro. We demonstrated that U50,488H inhibited VSMC calcification in a concentration-dependent manner. Moreover, U50,488H significantly inhibited osteogenic differentiation and ALP activity in VSMCs pretreated with ß-GP. Further studies confirmed that PFKFB3 expression, LDH level, and lactate content significantly increased during calcification of VSMCs; U50,488H reversed these changes. PHD2 expression showed the opposite trend compared to PFKFB3 expression. nor-BNI or 3-PO abolished U50,488H protective effects. Besides, U50,488H inhibited VSMC calcification in rat aortic rings ex vivo. Collectively, our experiments show that κ-opioid receptor activation inhibits VSMC calcification by reducing PFKFB3 expression and lactate content, providing a potential drug target and strategy for the clinical treatment of vascular calcification.


Assuntos
Ácido Láctico/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fosfofrutoquinase-2/metabolismo , Receptores Opioides kappa/metabolismo , Transdução de Sinais , Calcificação Vascular/metabolismo , Animais , Aorta/patologia , Diferenciação Celular/efeitos dos fármacos , Glicerofosfatos/farmacologia , Glicólise/efeitos dos fármacos , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Calcificação Vascular/patologia
5.
Aging (Albany NY) ; 13(10): 14416-14432, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021541

RESUMO

We investigated the role of microRNA (miR)-485 and its downstream signaling molecules on mediating epilepsy in cellular and rat models. We established a cellular epilepsy model by exposing hippocampal neurons to magnesium and a rat model by treating ICR mice with lithium chloride (127 mg/kg) and pilocarpine (30 mg/kg). We confirmed that miR-485 could bind and inhibit histone deacetylase 5 (HDAC5) and then measured expression of miR-485 and in mice and cells. Cells were transfected with overexpression or knockdown of miR-485, HDAC5, hypoxia-inducible factor-1alpha (HIF1α), or 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 enzyme (PFKFB3) to verify their roles in apoptosis, oxidative stress, and inflammation in epileptic hippocampal neurons. Binding relationship between miR-485, HDAC5, HIF1α, and PFKFB3 was verified. Oxidative stress and inflammation marker levels in epilepsy model mice were assessed. miR-485 was downregulated and HDAC5 was upregulated in cell and animal model of epilepsy. Seizure, neuronal apoptosis, oxidative stress (increased SOD and GSH-Px expression and decreased MDA and 8-OHdG expression) and inflammation (reduced IL-1ß, TNF-α, and IL-6 expression) were reduced by miR-485 in epileptic cells. HIF1α and PFKFB3 expression was reduced by HDAC5 knockdown in cells, which was recapitulated in vivo. Thus, miR-485 alleviates neuronal damage and epilepsy by inhibiting HDAC5, HIF1α, and PFKFB3.


Assuntos
Epilepsia/genética , Histona Desacetilases/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , MicroRNAs/metabolismo , Fosfofrutoquinase-2/genética , Animais , Apoptose/genética , Sequência de Bases , Modelos Animais de Doenças , Regulação para Baixo/genética , Hipocampo/patologia , Inflamação/genética , Inflamação/patologia , Masculino , Camundongos Endogâmicos ICR , MicroRNAs/genética , Neurônios/metabolismo , Estresse Oxidativo/genética , Fosfofrutoquinase-2/metabolismo , Ratos , Regulação para Cima/genética
6.
Int J Mol Sci ; 22(8)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33923905

RESUMO

Estrogen receptor (ER) activity mediates multiple physiological processes in the cardiovascular system. ERα and ERß are ligand-activated transcription factors of the nuclear hormone receptor superfamily, while the G protein-coupled estrogen receptor (GPER) mediates estrogenic signals by modulating non-nuclear second messengers, including activation of the MAP kinase signaling cascade. Membrane localizations of ERs are generally associated with rapid, non-genomic effects while nuclear localizations are associated with nuclear activities/transcriptional modulation of target genes. Gender dependence of endothelial biology, either through the action of sex hormones or sex chromosome-related factors, is becoming increasingly evident. Accordingly, cardiometabolic risk increases as women transition to menopause. Estrogen pathways control angiogenesis progression through complex mechanisms. The classic ERs have been acknowledged to function in mediating estrogen effects on glucose metabolism, but 17ß-estradiol also rapidly promotes endothelial glycolysis by increasing glucose transporter 1 (GLUT1) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) levels through GPER-dependent mechanisms. Estrogens alter monocyte and macrophage phenotype(s), and induce effects on other estrogen-responsive cell lineages (e.g., secretion of cytokines/chemokines/growth factors) that impact macrophage function. The pharmacological modulation of ERs for therapeutic purposes, however, is particularly challenging due to the lack of ER subtype selectivity of currently used agents. Identifying the determinants of biological responses to estrogenic agents at the vascular immune interface and developing targeted pharmacological interventions may result in novel improved therapeutic solutions.


Assuntos
Receptores de Estrogênio/metabolismo , Animais , Estrogênios/metabolismo , Estrogênios/farmacologia , Transportador de Glucose Tipo 1/metabolismo , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fosfofrutoquinase-2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
7.
Life Sci ; 276: 119412, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33774025

RESUMO

AIMS: The effects of PFKFB4 on glycolysis during the cancer progression has been investigated, while its role in glioma remains unclear. The present study evaluated the molecular mechanism of PFKFB4 in glycolysis of glioma progression. MATERIALS AND METHODS: The pan-cancer platform SangerBox was inquired to investigate the E2F2 expression in tumors. The E2F2 expression was studied by qRT-PCR and immunohistochemistry in collected glioma and normal brain tissues and by qRT-PCR and western blot in glioma cells. The relationship between the E2F2 expression in glioma tissues and patients' prognosis was analyzed. The cell malignant phenotype, glycolysis, growth and metastasis were examined by CCK-8, EdU, colony formation, flow cytometry, wound healing, Transwell assays, ELISA kits, and tumorigenesis and metastasis assays. Downstream targets of E2F2 were searched in hTFtarget, followed by pathway enrichment analysis. The expression of these targets and their correlation with E2F2 expression in gliomas were investigated through the GEPIA website. After ChIP and luciferase assays, the effect of the target on glioma was investigated. KEY FINDINGS: E2F2 was overexpressed in glioma patients and predicted poor prognoses. E2F2 promoted cell proliferation, colony formation, DNA synthesis, migration, invasion and glycolysis, and inhibited apoptosis. Meanwhile, inhibition of E2F2 suppressed the growth and metastasis of gliomas. E2F2 elevated the PFKFB4 expression transcriptionally by binding to its promoter and activated PI3K/AKT pathway. The promotion of glioma metastasis and glycolysis by E2F2 was mitigated by PFKFB4 knockdown. SIGNIFICANCE: E2F2-mediated transcriptional enhancement of PFKFB4 expression regulated the phosphorylation of PI3K/AKT to promote glioma malignancy progression.


Assuntos
Biomarcadores Tumorais/metabolismo , Fator de Transcrição E2F2/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfofrutoquinase-2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Fator de Transcrição E2F2/genética , Feminino , Glioma/genética , Glioma/metabolismo , Glicólise , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/genética , Fosfofrutoquinase-2/genética , Fosforilação , Prognóstico , Proteínas Proto-Oncogênicas c-akt/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Commun Biol ; 4(1): 188, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33580152

RESUMO

Hyperamylinemia induces amylin aggregation and toxicity in the pancreas and contributes to the development of type-2 diabetes (T2D). Cardiac amylin deposition in patients with obesity and T2D was found to accelerate heart dysfunction. Non-human primates (NHPs) have similar genetic, metabolic, and cardiovascular processes as humans. However, the underlying mechanisms of cardiac amylin in NHPs, particularly related to the hypoxia inducible factor (HIF)1α and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) signaling pathways, are unknown. Here, we demonstrate that in NHPs, amylin deposition in heart failure (HF) contributes to cardiac dysfunction via activation of HIF1α and PFKFB3 signaling. This was confirmed in two in vitro cardiomyocyte models. Furthermore, alterations of intracellular Ca2+, reactive oxygen species, mitochondrial function, and lactate levels were observed in amylin-treated cells. Our study demonstrates a pathological role for amylin in the activation of HIF1α and PFKFB3 signaling in NHPs with HF, establishing amylin as a promising target for heart disease patients.


Assuntos
Insuficiência Cardíaca/enzimologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Miocárdio/enzimologia , Fosfofrutoquinase-2/metabolismo , Animais , Apoptose , Sinalização do Cálcio , Células Cultivadas , Modelos Animais de Doenças , Feminino , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Macaca fascicularis , Masculino , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/patologia , Miocárdio/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Volume Sistólico , Função Ventricular Esquerda , Pressão Ventricular
9.
Biochem Biophys Res Commun ; 544: 52-59, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33516882

RESUMO

Dental pulp stem cells (DPSCs) can differentiate into diverse cell lineages, including odontogenic cells that are responsible for dentin formation, which is important in pulp repair and tooth regeneration. While glycolysis plays a central role in various cellular activities in both physiological and pathological conditions, its role and regulation in odontogenic differentiation are unknown. Here, we show that aerobic glycolysis is induced during odontoblastic differentiation from human DPSCs. Importantly, we demonstrate that during odontoblastic differentiation, protein expression levels of phosphofructokinase 1 muscle isoform (PFKM) and PFK2, but not other glycolytic enzymes, are mainly upregulated by AKT activation, resulting in increased total PFK enzyme activity. Increased PFK activity is essential to enhance aerobic glycolysis, which plays an important role in the odontoblastic differentiation of human DPSCs. These findings underscore that PFK activation-induced aerobic glycolysis accompanies, and participates in, human DPSCs differentiation into odontogenic lineage, and could play a role in the regulation of dental pulp repair.


Assuntos
Diferenciação Celular , Polpa Dentária/citologia , Odontogênese , Fosfofrutoquinase-1 Muscular/metabolismo , Fosfofrutoquinase-2/metabolismo , Células-Tronco/citologia , Proliferação de Células , Células Cultivadas , Polpa Dentária/metabolismo , Humanos , Transdução de Sinais , Células-Tronco/metabolismo
10.
FASEB J ; 35(2): e21343, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33508151

RESUMO

Most physiological processes in mammals are subjected to daily oscillations that are governed by a circadian system. The circadian rhythm orchestrates metabolic pathways in a time-dependent manner and loss of circadian timekeeping has been associated with cellular and system-wide alterations in metabolism, redox homeostasis, and inflammation. Here, we investigated the expression of clock and clock-controlled genes in multiple tissues (suprachiasmatic nucleus, spinal cord, gastrocnemius muscle, and liver) from mutant hSOD1-linked amyotrophic lateral sclerosis (ALS) mouse models. We identified tissue-specific changes in the relative expression, as well as altered daily expression patterns, of clock genes, sirtuins (Sirt1, Sirt3, and Sirt6), metabolic enzymes (Pfkfb3, Cpt1, and Nampt), and redox regulators (Nrf2, G6pd, and Pgd). In addition, astrocytes transdifferentiated from induced pluripotent stem cells from SOD1-linked and FUS RNA binding protein-linked ALS patients also displayed altered expression of clock genes. Overall, our results raise the possibility of disrupted cross-talk between the suprachiasmatic nucleus and peripheral tissues in hSOD1G93A mice, preventing proper peripheral clock regulation and synchronization. Since these changes were observed in symptomatic mice, it remains unclear whether this dysregulation directly drives or it is a consequence of the degenerative process. However, because metabolism and redox homeostasis are intimately entangled with circadian rhythms, our data suggest that altered expression of clock genes may contribute to metabolic and redox impairment in ALS. Since circadian dyssynchrony can be rescued, these results provide the groundwork for potential disease-modifying interventions.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteínas CLOCK/metabolismo , Superóxido Dismutase-1/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Astrócitos/metabolismo , Proteínas CLOCK/genética , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Fosfogluconato Desidrogenase/genética , Fosfogluconato Desidrogenase/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismo
11.
Lab Invest ; 101(3): 328-340, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33462362

RESUMO

Obesity-associated inflammation in white adipose tissue (WAT) is a causal factor of systemic insulin resistance; however, precisely how immune cells regulate WAT inflammation in relation to systemic insulin resistance remains to be elucidated. The present study examined a role for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in hematopoietic cells in regulating WAT inflammation and systemic insulin sensitivity. Male C57BL/6J mice were fed a high-fat diet (HFD) or low-fat diet (LFD) for 12 weeks and examined for WAT inducible 6-phosphofructo-2-kinase (iPFK2) content, while additional HFD-fed mice were treated with rosiglitazone and examined for PFKFB3 mRNAs in WAT stromal vascular cells (SVC). Also, chimeric mice in which PFKFB3 was disrupted only in hematopoietic cells and control chimeric mice were also fed an HFD and examined for HFD-induced WAT inflammation and systemic insulin resistance. In vitro, adipocytes were co-cultured with bone marrow-derived macrophages and examined for adipocyte proinflammatory responses and insulin signaling. Compared with their respective levels in controls, WAT iPFK2 amount in HFD-fed mice and WAT SVC PFKFB3 mRNAs in rosiglitazone-treated mice were significantly increased. When the inflammatory responses were analyzed, peritoneal macrophages from PFKFB3-disrputed mice revealed increased proinflammatory activation and decreased anti-inflammatory activation compared with control macrophages. At the whole animal level, hematopoietic cell-specific PFKFB3 disruption enhanced the effects of HFD feeding on promoting WAT inflammation, impairing WAT insulin signaling, and increasing systemic insulin resistance. In vitro, adipocytes co-cultured with PFKFB3-disrupted macrophages revealed increased proinflammatory responses and decreased insulin signaling compared with adipocytes co-cultured with control macrophages. These results suggest that PFKFB3 disruption in hematopoietic cells only exacerbates HFD-induced WAT inflammation and systemic insulin resistance.


Assuntos
Tecido Adiposo Branco/metabolismo , Inflamação/metabolismo , Resistência à Insulina/fisiologia , Obesidade/metabolismo , Fosfofrutoquinase-2/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo Branco/citologia , Animais , Células Cultivadas , Dieta com Restrição de Gorduras , Dieta Hiperlipídica , Modelos Animais de Doenças , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Transdução de Sinais
12.
Cells ; 9(12)2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33302475

RESUMO

Phenanthriplatin is a new monofunctional platinum(II) complex that binds only one strand of DNA and acts by blocking gene transcription, but its effect on gene regulation has not been characterized relative to the traditional platinum-based complex, cisplatin. A549 non-small cell lung cancer and IMR90 lung fibroblast cells were treated with cisplatin, phenanthriplatin, or a control and then their RNA transcripts were subjected to next generation sequencing analysis. DESeq2 and CuffDiff2 were used to identify up- and downregulated genes and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used to identify pathways and functions. We found that phenanthriplatin may regulate the genes GPRC5a, TFF1, and TNFRSF10D, which act through p53 to control apoptosis, differently or to a greater extent than cisplatin, and that it, unlike cisplatin, could upregulate ATP5MD, a gene which signals through the Wnt/ß catenin pathway. Furthermore, phenanthriplatin caused unique or enhanced effects compared to cisplatin on genes regulating the cytoskeleton, cell migration, and proliferation, e.g., AGAP1, DIAPH2, GDF15, and THSD1 (p < 0.05; q < 0.05). Phenanthriplatin may modulate some oncogenes differently than cisplatin potentially leading to improved clinical outcome, but this monofunctional complex should be carefully matched with cancer gene data to be successfully applied in chemotherapy.


Assuntos
Cisplatino/farmacologia , Fibroblastos/efeitos dos fármacos , Compostos Organoplatínicos/farmacologia , Fenantridinas/farmacologia , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Platina/química , Análise de Sequência de RNA , Regulação para Cima/efeitos dos fármacos
13.
Sci Rep ; 10(1): 16754, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028909

RESUMO

L-arginine/NOS/NO signaling pathway plays a critical role in controlling variety of vascular diseases. However, whether NOS inhibition by L-NAME suppresses late embryonic development is undefined. The aim of this study is to determine whether NOS inhibition by L-NAME is critical for late embryonic rat hind limb development. The pregnant rat at E13.5 administrated L-NAME by consecutive intraperitoneal injection. The embryos been harvested from E16.5 to E 20.5. Hematoxylin and Eosin Staining, Immunofluorescence and Immunohistochemistry performed to determine hind limb Vasculogenesis, HUVEC culture, Adenoviral PFKFB3 infection, Real time PCR and western blot were performed to determine whether L-arginine/NOS/NO pathway controlling late embryonic hind limb development through PFKFB3 mediated angiogenetic pathway. NOS inhibition by L-NAME resulting in late embryonic hind limb developmental defects characterized by severe hemorrhage. The in vivo studies showed that NOS inhibition strongly suppressed hind limb angiogenetic remodeling by impairing differentiation of endothelial cells and smooth muscle cells, and extracellular matrix synthesis. For underlie mechanism, our studies indicated that L-NAME treatment dramatically suppresses PFKFB3 expression in hematopoietic progenitor cells, tubulogenetic endothelial cells and smooth muscle cells. Knockdown of PFKFB3 dramatically inhibits the expression of angiogenetic genes, as well as tubulogenesis and extracellular matrix related genes. Taken together, our data in this study demonstrated that L-arginine-eNOS-NO pathway is important for rat hind limb development during late embryonic stage. This could be both a useful animal model and a promising therapeutic treatment for defects of late embryonic developmental hind limbs.


Assuntos
Inibidores Enzimáticos/farmacologia , Membro Posterior/embriologia , NG-Nitroarginina Metil Éster/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Fosfofrutoquinase-2/metabolismo , Animais , Feminino , Membro Posterior/irrigação sanguínea , Gravidez , Ratos , Fluxo Sanguíneo Regional/efeitos dos fármacos
14.
Int J Mol Sci ; 21(20)2020 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-33066028

RESUMO

Neural tube closure is a critical early step in central nervous system development that requires precise control of metabolism to ensure proper cellular proliferation and differentiation. Dysregulation of glucose metabolism during pregnancy has been associated with neural tube closure defects (NTDs) in humans suggesting that the developing neuroepithelium is particularly sensitive to metabolic changes. However, it remains unclear how metabolic pathways are regulated during neurulation. Here, we used single-cell mRNA-sequencing to analyze expression of genes involved in metabolism of carbon, fats, vitamins, and antioxidants during neurulation in mice and identify a coupling of glycolysis and cellular proliferation to ensure proper neural tube closure. Using loss of miR-302 as a genetic model of cranial NTD, we identify misregulated metabolic pathways and find a significant upregulation of glycolysis genes in embryos with NTD. These findings were validated using mass spectrometry-based metabolite profiling, which identified increased glycolytic and decreased lipid metabolites, consistent with a rewiring of central carbon traffic following loss of miR-302. Predicted miR-302 targets Pfkp, Pfkfb3, and Hk1 are significantly upregulated upon NTD resulting in increased glycolytic flux, a shortened cell cycle, and increased proliferation. Our findings establish a critical role for miR-302 in coordinating the metabolic landscape of neural tube closure.


Assuntos
Ciclo Celular , Glicólise , MicroRNAs/metabolismo , Tubo Neural/metabolismo , Neurulação , Animais , Células Cultivadas , Hexoquinase/genética , Hexoquinase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Tubo Neural/embriologia , Fosfofrutoquinase-1 Tipo C/genética , Fosfofrutoquinase-1 Tipo C/metabolismo , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo
15.
Sci Rep ; 10(1): 14531, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32884050

RESUMO

Fatty acid oxidation is the major energy pathway used by the kidney, although glycolysis becomes more important in the low oxygen environment of the medulla. Fatty acid oxidation appears to be reduced in renal fibrosis, and drugs that reverse this improve fibrosis. Expression of glycolytic genes is more variable, but some studies have shown that inhibiting glycolysis reduces renal fibrosis. To address the role of glycolysis in renal fibrosis, we have used a genetic approach. The crucial control point in the rate of glycolysis is 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase. Phosphorylation of the kidney isoform, PFKFB2, on residues Ser468 and Ser485 stimulates glycolysis and is the most important mechanism regulating glycolysis. We generated transgenic mice with inactivating mutations of Ser468 and Ser485 in PFKFB2 (PFKFB2 KI mice). These mutations were associated with a reduced ability to increase glycolysis in primary cultures of renal tubular cells from PFKFB2 KI mice compared to WT cells. This was associated in PFKFB2 KI mice with increased renal fibrosis, which was more severe in the unilaternal ureteric obstruction (UUO) model compared with the folic acid nephropathy (FAN) model. These studies show that phosphorylation of PFKFB2 is important in limiting renal fibrosis after injury, indicating that the ability to regulate and maintain adequate glycolysis in the kidney is crucial for renal homeostasis. The changes were most marked in the UUO model, probably reflecting a greater effect on distal renal tubules and the greater importance of glycolysis in the distal nephron.


Assuntos
Fibrose/metabolismo , Fibrose/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Fosfofrutoquinase-2/metabolismo , Fosforilação/fisiologia , Animais , Western Blotting , Células Cultivadas , Fibrose/genética , Rim/metabolismo , Rim/patologia , Nefropatias/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Fosfofrutoquinase-2/genética , Fosforilação/genética
16.
Cell Transplant ; 29: 963689720950226, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841050

RESUMO

Apoptosis is a vital pathological factor that accounts for the poor prognosis of traumatic spinal cord injury (t-SCI). The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) is a critical regulator for energy metabolism and proven to have antiapoptotic effects. This study aimed to investigate the neuroprotective role of PFKFB3 in t-SCI. A compressive clip was introduced to establish the t-SCI model. Herein, we identified that PFKFB3 was extensively distributed in neurons, and PFKFB3 levels significantly increased and peaked 24 h after t-SCI. Additionally, knockdown of PFKFB3 inhibited glycolysis, accompanied by aggravated neuronal apoptosis and white matter injury, while pharmacological activation of PFKFB3 with meclizine significantly enhanced glycolysis, attenuated t-SCI-induced spinal cord injury, and alleviated neurological impairment. The PFKFB3 agonist, meclizine, activated cyclin-dependent kinase 1 (CDK1) and promoted the phosphorylation of p27, ultimately suppressing neuronal apoptosis. However, the neuroprotective effects of meclizine against t-SCI were abolished by the CDK1 antagonist, RO3306. In summary, our data demonstrated that PFKFB3 contributes robust neuroprotection against t-SCI by enhancing glycolysis and modulating CDK1-related antiapoptotic signals. Moreover, targeting PFKFB3 may be a novel and promising therapeutic strategy for t-SCI.


Assuntos
Apoptose , Proteína Quinase CDC2/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Glicólise , Neurônios/patologia , Fosfofrutoquinase-2/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Animais , Apoptose/efeitos dos fármacos , Frutosedifosfatos/metabolismo , Técnicas de Silenciamento de Genes , Glicólise/efeitos dos fármacos , Ácido Láctico/metabolismo , Masculino , Meclizina/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Modelos Biológicos , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Quinolinas/farmacologia , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Medula Espinal/ultraestrutura , Traumatismos da Medula Espinal/fisiopatologia , Tiazóis/farmacologia , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos , Substância Branca/lesões , Substância Branca/patologia
17.
Life Sci ; 259: 118215, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32768579

RESUMO

AIMS: Infantile hemangioma (IH) is one of the most common tumors in infancy, which etiology and pathogenesis has not been fully elucidated, hypoxia and abnormal glucose metabolism is regarded as critical pathogenic factors. This study investigated the expression and function of glycolysis-associated molecules (GLUT1, HK2, PFKFB3, PKM2, and LDHA) under normoxic and hypoxic conditions to further understand the pathogenesis of IH. MAIN METHODS: Hemangioma-derived endothelial cells (HemECs) were isolated from proliferating phase infantile hemangiomas and identified by immunofluorescence. HemECs and human umbilical vein endothelial cells (HUVECs) were cultured under normoxic and hypoxic conditions. RNA and protein expression of glycolysis-associated molecules were analyzed by quantitative real-time RT-PCR, western blotting, and immunohistochemistry. Glucose consumption, ATP production and lactate production were measured. Glycolysis-associated molecules were inhibited by WZB117, 3BP, 3PO, SKN, and GSK 2837808A and the resulting effects on HemECs proliferation, migration, and tube formation were quantified. KEY FINDINGS: Glycolysis-associated molecules were highly expressed at both mRNA and protein levels in HemECs compared with HUVECs (P < 0.05). Glucose consumption and ATP production were higher in HemECs than in HUVECs, while lactate production in HemECs was lower than in HUVECs (P < 0.05). Inhibition of some glycolysis-associated molecules reduced the proliferation, migration, and tube formation capacity of HemECs (P < 0.05). SIGNIFICANCE: Our study revealed that glycolysis-associated molecules were highly expressed in IH. Glucose metabolismin HemECs differed from normal endothelial cells. Altering the expression of glycolysis-associated molecules may influence the phenotype of HemECs and provide new therapeutic approaches to the successful treatment of IH.


Assuntos
Glicólise/fisiologia , Hemangioma/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , China , Feminino , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Glicólise/genética , Hemangioma/fisiopatologia , Hexoquinase/genética , Hexoquinase/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipóxia , Lactente , Recém-Nascido , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Masculino , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/genética
18.
Sci Transl Med ; 12(555)2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32759274

RESUMO

The coordination of metabolic signals among different cellular components in pathological retinal angiogenesis is poorly understood. Here, we showed that in the pathological angiogenic vascular niche, retinal myeloid cells, particularly macrophages/microglia that are spatially adjacent to endothelial cells (ECs), are highly glycolytic. We refer to these macrophages/microglia that exhibit a unique angiogenic phenotype with increased expression of both M1 and M2 markers and enhanced production of both proinflammatory and proangiogenic cytokines as pathological retinal angiogenesis-associated glycolytic macrophages/microglia (PRAGMs). The phenotype of PRAGMs was recapitulated in bone marrow-derived macrophages or retinal microglia stimulated by lactate that was produced by hypoxic retinal ECs. Knockout of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFKFB3; Pfkfb3 for rodents), a glycolytic activator in myeloid cells, impaired the ability of macrophages/microglia to acquire an angiogenic phenotype, rendering them unable to promote EC proliferation and sprouting and pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Mechanistically, hyperglycolytic macrophages/microglia produced large amount of acetyl-coenzyme A, leading to histone acetylation and PRAGM-related gene induction, thus reprogramming macrophages/microglia into an angiogenic phenotype. These findings reveal a critical role of glycolytic metabolites as initiators of reciprocal activation of macrophages/microglia and ECs in the retinal angiogenic niche and suggest that strategies targeting the metabolic communication between these cell types may be efficacious in the treatment of pathological retinal angiogenesis.


Assuntos
Células Endoteliais , Glicólise , Animais , Células Endoteliais/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Fosfofrutoquinase-2/metabolismo
19.
FASEB J ; 34(9): 12768-12784, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32757462

RESUMO

Few studies have explored the mechanisms coupling estrogen signals to metabolic demand in endothelial cells. We recently showed that 17ß-estradiol (E2) triggers angiogenesis via the membrane G-protein coupled estrogen receptor (GPER) and the key glycolytic protein PFKFB3 as a downstream effector. We herein investigated whether estrogenic agents regulate the stability and/or degradation of glycolytic proteins in human umbilical vein endothelial cells (HUVECs). Similarly to E2, the GPER selective agonist G1 rapidly increased PFKFB3 protein amounts, without affecting mRNA levels. In the presence of cycloheximide, E2 and G1 treatment counteracted PFKFB3 degradation over time, whereas E2-induced PFKFB3 stabilization was abolished by the GPER antagonist G15. Inhibitors of selective SCF E3 ubiquitin ligase (SMER-3) and proteasome (MG132) rapidly increased PFKFB3 protein levels. Accordingly, ubiquitin-bound PFKFB3 was lower in E2- or G1-treated HUVECs. Both agents increased deubiquitinase USP19 levels through GPER signaling. Notably, USP 19 siRNA decreased PFKFB3 levels and abolished E2- and G1-mediated HUVEC tubularization. Finally, E2 and G1 treatments rapidly enhanced glucose transporter GLUT1 levels via GPER independent of transcriptional activation. These findings provide new evidence on mechanisms coupling estrogen signals with the glycolytic program in endothelium and unravel the role of USP19 as a target of the pro-angiogenic effect of estrogenic agents.


Assuntos
Endopeptidases/metabolismo , Estradiol/farmacologia , Transportador de Glucose Tipo 1/metabolismo , Fosfofrutoquinase-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos
20.
Theranostics ; 10(16): 7245-7259, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32641990

RESUMO

Rationale: Tumor vascular normalization (TVN) is emerging to enhance the efficacy of anticancer treatment in many cancers including glioblastoma (GBM). However, a common and severe challenge being currently faced is the transient TVN effect, hampering the sustained administration of anticancer therapy during TVN window. Additionally, the lack of non-contrast agent-based imaging biomarkers to monitor TVN process postpones the clinical translation of TVN strategy. In this study, we investigated whether dual inhibition of VEGF and the glycolytic activator PFKFB3 could reinforce the TVN effect in GBM. Dynamic contrast-enhanced-magnetic resonance imaging (DCE-MRI) and intravoxel incoherent motion (IVIM)-MRI were performed to monitor TVN process and to identify whether IVIM-MRI is a candidate or complementary imaging biomarker for monitoring TVN window without exogenous contrast agent administration. Methods: Patient-derived orthotopic GBM xenografts in mice were established and treated with bevacizumab (BEV), 3PO (PFKFB3 inhibitor), BEV+3PO dual therapy, or saline. The vascular morphology, tumor hypoxia, and lactate level were evaluated before and at different time points after treatments. Doxorubicin was used to evaluate chemotherapeutic efficacy and drug delivery. Microarray of angiogenesis cytokines and western blotting were conducted to characterize post-treatment molecular profiling. TVN process was monitored by DCE- and IVIM-MRI. Correlation analysis of pathological indicators and MRI parameters was further analyzed. Results: Dual therapy extended survival and delayed tumor growth over each therapy alone, concomitant with a decrease of cell proliferation and an increase of cell apoptosis. The dual therapy reinforces TVN effect, thereby alleviating tumor hypoxia, reducing lactate production, and improving the efficacy and delivery of doxorubicin. Mechanistically, several angiogenic cytokines and pathways were downregulated after dual therapy. Notably, dual therapy inhibited Tie1 expression, the key regulator of TVN, in both endothelial cells and tumor cells. DCE- and IVIM-MRI data showed that dual therapy induced a more homogenous and prominent TVN effect characterized by improved vascular function in tumor core and tumor rim. Correlation analysis revealed that IVIM-MRI parameter D * had better correlations with TVN pathological indicators compared with the DCE-MRI parameter K trans. Conclusions: Our results propose a rationale to overcome the current limitation of BEV monotherapy by integrating the synergistic effects of VEGF and PFKFB3 blockade to enhance chemotherapy efficacy through a sustained TVN effect. Moreover, we unveil IVIM-MRI parameter D * has much potential as a complementary imaging biomarker to monitor TVN window more precisely without exogenous contrast agent injection.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Neovascularização Patológica/diagnóstico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Bevacizumab/farmacologia , Bevacizumab/uso terapêutico , Encéfalo/irrigação sanguínea , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Glioblastoma/irrigação sanguínea , Glioblastoma/diagnóstico , Glioblastoma/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Ácido Láctico/análise , Ácido Láctico/metabolismo , Masculino , Camundongos , Imageamento por Ressonância Magnética Multiparamétrica , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Fosfofrutoquinase-2/antagonistas & inibidores , Fosfofrutoquinase-2/metabolismo , Piridinas/farmacologia , Piridinas/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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