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1.
Methods Mol Biol ; 2326: 33-46, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34097259

RESUMO

Environmental pollutants inevitably exert adverse effects on humans and other species. Quick identification and in-depth characterization of the pollutants are requisite objectives for clinicians and environmental health scientists. The nematode Caenorhabditis elegans has been utilized as a model organism for toxicity evaluation of environmental pollutants, due to its transparency, short lifespan, entire genome sequencing, and economical characteristics. However, few researchers have systematically addressed mitochondrial toxicity in response to toxicants, despite the critical role mitochondria play in energy production and respiration, as well as the generation of reactive oxygen species. Mitochondria are vulnerable to environmental pollutants, and their dysfunction contributes to cellular damage and toxicity in plethora of diseases. Here, we describe methods in step-by-step for mitochondrial toxicity evaluation in response to pollutants, including exposure of C. elegans to toxicants, mitochondrial ROS detection, mitochondrial morphology analysis, mitochondrial function analysis, such as ATP production and oxygen consumption, and gene expression studies, with the application of corresponding genetically modified strains.


Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Mitocôndrias/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Expressão Gênica/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Consumo de Oxigênio/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Testes de Toxicidade/métodos
2.
Methods Mol Biol ; 2277: 391-403, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080164

RESUMO

Cellular metabolism contributes to cell fate decisions. Bioenergetic profiling can therefore provide considerable insights into cellular identity and specification. Given the current importance of human pluripotent stem cells (hPSCs) for biomedical applications, assessing the bioenergetic properties of hPSCs and derivatives can unveil relevant mechanisms in the context of development biology and molecular disease modeling. Here, we describe a method to facilitate bioenergetic profiling of hPSCs in a reproducible and scalable manner. After simultaneous assessment of mitochondrial respiration and glycolytic capacity using Seahorse XFe96 Analyzer, we measure lactate concentration in the cellular media. Finally, we normalize the values based on DNA amount. We describe the procedures with specific requirements related to hPSCs . However, the same protocol can be easily adapted to other cell types, including differentiated progenies from hPSCs .


Assuntos
Mitocôndrias/metabolismo , Biologia Molecular/métodos , Células-Tronco Pluripotentes/metabolismo , Antimicina A/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Técnicas de Cultura de Células/métodos , DNA/análise , Metabolismo Energético/efeitos dos fármacos , Humanos , Ácido Láctico/análise , Mitocôndrias/efeitos dos fármacos , Oligomicinas/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Rotenona/farmacologia
3.
Nat Commun ; 12(1): 3669, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135327

RESUMO

Human rhinoviruses (HRV) are common cold viruses associated with exacerbations of lower airways diseases. Although viral induced epithelial damage mediates inflammation, the molecular mechanisms responsible for airway epithelial damage and dysfunction remain undefined. Using experimental HRV infection studies in highly differentiated human bronchial epithelial cells grown at air-liquid interface (ALI), we examine the links between viral host defense, cellular metabolism, and epithelial barrier function. We observe that early HRV-C15 infection induces a transitory barrier-protective metabolic state characterized by glycolysis that ultimately becomes exhausted as the infection progresses and leads to cellular damage. Pharmacological promotion of glycolysis induces ROS-dependent upregulation of the mitochondrial metabolic regulator, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), thereby restoring epithelial barrier function, improving viral defense, and attenuating disease pathology. Therefore, PGC-1α regulates a metabolic pathway essential to host defense that can be therapeutically targeted to rescue airway epithelial barrier dysfunction and potentially prevent severe respiratory complications or secondary bacterial infections.


Assuntos
Antivirais/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Mucosa Respiratória/metabolismo , Rhinovirus/fisiologia , Complexos de ATP Sintetase/antagonistas & inibidores , Complexos de ATP Sintetase/metabolismo , Antivirais/farmacologia , Técnicas de Cultura de Células , Citoesqueleto/metabolismo , Células Epiteliais , Ácidos Graxos/biossíntese , Glicólise , Humanos , Redes e Vias Metabólicas , Mitocôndrias/metabolismo , Oligomicinas/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Infecções por Picornaviridae/virologia , Espécies Reativas de Oxigênio/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Replicação Viral/efeitos dos fármacos
4.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063028

RESUMO

Myocardial infarction is a leading cause for morbidity and mortality worldwide. The only viable treatment for the ischemic insult is timely reperfusion, which further exacerbates myocardial injury. Maintaining mitochondrial function is crucial in preserving cardiomyocyte function in ischemia reperfusion (IR) injury. Poloxamer (P) 188 has been shown to improve cardiac IR injury by improving cellular and mitochondrial function. The aim of this study was to show if P188 postconditioning has direct protective effects on mitochondrial function in the heart. Langendorff prepared rat hearts were subjected to IR injury ex-vivo and reperfused for 10 min with 1 mM P188 vs. vehicle. Cardiac mitochondria were isolated with 1 mM P188 vs. 1 mM polyethylene glycol (PEG) vs. vehicle by differential centrifugation. Mitochondrial function was assessed by adenosine triphosphate synthesis, oxygen consumption, and calcium retention capacity. Mitochondrial function decreased significantly after ischemia and showed mild improvement with reperfusion. P188 did not improve mitochondrial function in the ex-vivo heart, and neither further P188 nor PEG induced direct mitochondrial protection after IR injury in this model.


Assuntos
Pós-Condicionamento Isquêmico , Mitocôndrias Cardíacas/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , Poloxâmero/farmacologia , Trifosfato de Adenosina/biossíntese , Animais , Cálcio/metabolismo , Respiração Celular/efeitos dos fármacos , Complexo II de Transporte de Elétrons/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Ratos Sprague-Dawley
5.
Nat Commun ; 12(1): 2804, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990571

RESUMO

Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Mitocôndrias/metabolismo , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/fisiologia , Feminino , Proteínas de Choque Térmico HSP40/deficiência , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos
6.
Am Heart J ; 239: 1-10, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33992607

RESUMO

Sacubitril/valsartan reduces mortality in patients with heart failure with reduced ejection fraction (HFrEF) when compared with enalapril. However, it is unknown the effect of both treatments on exercise capacity. We compared sacubitril/valsartan versus enalapril in patients with HFrEF based on peak oxygen consumption (VO2) and 6-minute walk test (6-MWT). METHODS: We included 52 participants with HFrEF with a left ventricular ejection fraction <40% to receive either sacubitril/valsartan (target dose of 400 mg daily) or enalapril (target dose of 40 mg daily). Peak VO2 was measured by using cardiopulmonary exercise testing. Six-minute walk test was also performed. RESULTS: At 12 weeks, the sacubitril/valsartan (mean dose 382.6 ± 57.6 mg daily) group had increased peak VO2 of 13.1% (19.35 ± 0.99 to 21.89 ± 1.04 mL/kg/min) and enalapril (mean dose 34.4 ± 9.2 mg daily) 5.6% (18.58 ± 1.19 to 19.62 ± 1.25 mL/kg/min). However, no difference was found between groups (P = .332 interaction). At 24 weeks, peak VO2 increased 13.5% (19.35 ± 0.99 to 21.96 ± 0.98 mL/kg/min) and 12.0% (18.58 ± 1.19 to 20.82 ± 1.18 mL/kg/min) in sacubitril/valsartan (mean dose 400 ± 0 mg daily) and enalapril (mean dose 32.7 ± 11.0 mg daily), respectively. However, no differences were found between groups (P= .332 interaction). At 12 weeks, 6-MWT increased in both groups (sacubitril/valsartan: 459 ± 18 to 488 ± 17 meters [6.3%] and enalapril: 443 ± 22 to 477 ± 21 meters [7.7%]). At 24 weeks, sacubitril/valsartan increased 18.3% from baseline (543 ± 26 meters) and enalapril decreased slightly to 6.8% (473 ± 31 meters), but no differences existed between groups (P= .257 interaction). CONCLUSIONS: Compared to enalapril, sacubitril/valsartan did not substantially improve peak VO2 or 6-MWT after 12 or 24 weeks in participants with HFrEF. (NEPRIExTol-HF Trial, ClinicalTrials.gov number, NCT03190304).


Assuntos
Aminobutiratos , Compostos de Bifenilo , Enalapril , Teste de Esforço , Tolerância ao Exercício/efeitos dos fármacos , Insuficiência Cardíaca , Valsartana , Disfunção Ventricular Esquerda , Aminobutiratos/administração & dosagem , Aminobutiratos/efeitos adversos , Antagonistas de Receptores de Angiotensina/administração & dosagem , Antagonistas de Receptores de Angiotensina/efeitos adversos , Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Inibidores da Enzima Conversora de Angiotensina/efeitos adversos , Compostos de Bifenilo/administração & dosagem , Compostos de Bifenilo/efeitos adversos , Método Duplo-Cego , Combinação de Medicamentos , Monitoramento de Medicamentos/métodos , Enalapril/administração & dosagem , Enalapril/efeitos adversos , Teste de Esforço/efeitos dos fármacos , Teste de Esforço/métodos , Feminino , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Consumo de Oxigênio/efeitos dos fármacos , Volume Sistólico , Valsartana/administração & dosagem , Valsartana/efeitos adversos , Disfunção Ventricular Esquerda/diagnóstico , Disfunção Ventricular Esquerda/fisiopatologia , Teste de Caminhada/métodos
7.
Am J Physiol Cell Physiol ; 321(1): C147-C157, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34038242

RESUMO

Many cancer cells, regardless of their tissue origin or genetic landscape, have increased expression or activity of the plasma membrane Na-H exchanger NHE1 and a higher intracellular pH (pHi) compared with untransformed cells. A current perspective that remains to be validated is that increased NHE1 activity and pHi enable a Warburg-like metabolic reprogramming of increased glycolysis and decreased mitochondrial oxidative phosphorylation. We tested this perspective and find it is not accurate for clonal pancreatic and breast cancer cells. Using the pharmacological reagent ethyl isopropyl amiloride (EIPA) to inhibit NHE1 activity and decrease pHi, we observe no change in glycolysis, as indicated by secreted lactate and intracellular pyruvate, despite confirming increased activity of the glycolytic enzyme phosphofructokinase-1 at higher pH. Also, in contrast to predictions, we find a significant decrease in oxidative phosphorylation with EIPA, as indicated by oxygen consumption rate (OCR). Decreased OCR with EIPA is not associated with changes in pathways that fuel oxidative phosphorylation or with mitochondrial membrane potential but occurs with a change in mitochondrial dynamics that includes a significant increase in elongated mitochondrial networks, suggesting increased fusion. These findings conflict with current paradigms on increased pHi inhibiting oxidative phosphorylation and increased oxidative phosphorylation being associated with mitochondrial fusion. Moreover, these findings raise questions on the suggested use of EIPA-like compounds to limit metabolic reprogramming in cancer cells.


Assuntos
Amilorida/análogos & derivados , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Dinâmica Mitocondrial/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Trocador 1 de Sódio-Hidrogênio/genética , Amilorida/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Células Clonais , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica , Glicólise/genética , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Fosfofrutoquinase-1/genética , Fosfofrutoquinase-1/metabolismo , Ácido Pirúvico/metabolismo , Trocador 1 de Sódio-Hidrogênio/antagonistas & inibidores , Trocador 1 de Sódio-Hidrogênio/metabolismo
8.
J Biol Chem ; 297(1): 100830, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34048714

RESUMO

Dietary lipid composition has been shown to impact brain morphology, brain development, and neurologic function. However, how diet uniquely regulates brain lipid homeostasis compared with lipid homeostasis in peripheral tissues remains largely uncharacterized. To evaluate the lipid response to dietary changes in the brain, we assessed actively translating mRNAs in astrocytes and neurons across multiple diets. From this data, ethanolamine phosphate phospholyase (Etnppl) was identified as an astrocyte-specific fasting-induced gene. Etnppl catabolizes phosphoethanolamine (PEtN), a prominent headgroup precursor in phosphatidylethanolamine (PE) also found in other classes of neurologically relevant lipid species. Altered Etnppl expression has also previously been associated with humans with mood disorders. We evaluated the relevance of Etnppl in maintaining brain lipid homeostasis by characterizing Etnppl across development and in coregulation with PEtN-relevant genes, as well as determining the impact to the brain lipidome after Etnppl loss. We found that Etnppl expression dramatically increased during a critical window of early brain development in mice and was also induced by glucocorticoids. Using a constitutive knockout of Etnppl (EtnpplKO), we did not observe robust changes in expression of PEtN-related genes. However, loss of Etnppl altered the phospholipid profile in the brain, resulting in increased total abundance of PE and in polyunsaturated fatty acids within PE and phosphatidylcholine species in the brain. Together, these data suggest that brain phospholipids are regulated by the phospholyase action of the enzyme Etnppl, which is induced by dietary fasting in astrocytes.


Assuntos
Astrócitos/metabolismo , Etanolaminas/metabolismo , Homeostase , Metabolismo dos Lipídeos , Fósforo-Oxigênio Liases/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Sistema Nervoso Central/citologia , Dieta , Jejum , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glucocorticoides/farmacologia , Homeostase/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Camundongos , Oxirredução , Consumo de Oxigênio/efeitos dos fármacos , Fosfolipídeos/metabolismo , Receptores de Glucocorticoides/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Especificidade por Substrato/efeitos dos fármacos
9.
Cancer Sci ; 112(7): 2652-2663, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33934440

RESUMO

Mitochondria are key cytoplasmic organelles. Their activation is critical for the generation of T cell proliferation and cytotoxicity. Exhausted tumor-infiltrating T cells show a decreased mitochondrial function and mass. 5-Aminolevulinic acid (5-ALA), a natural amino acid that is only produced in the mitochondria, has been shown to influence metabolic functions. We hypothesized that 5-ALA with sodium ferrous citrate (SFC) might provide metabolic support for tumor-infiltrating T cells. In a mouse melanoma model, we found that 5-ALA/SFC with a programmed cell death-ligand 1 (PD-L1) blocking Ab synergized tumor regression. After treatment with 5-ALA/SFC and anti-PD-L1 Ab, tumor infiltrating lymphocytes (TILs) were not only competent for the production of cytolytic particles and cytokines (granzyme B, interleukin-2, and γ-interferon) but also showed enhanced Ki-67 activity (a proliferation marker). The number of activated T cells (PD-1+ Tim-3- ) was also significantly increased. Furthermore, we found that 5-ALA/SFC activated the mitochondrial functions, including the oxygen consumption rate, ATP level, and complex V expression. The mRNA levels of Nrf-2, HO-1, Sirt-1, and PGC-1α and the protein levels of Sirt-1 were upregulated by treatment with 5-ALA/SFC. Taken together, our findings revealed that 5-ALA/SFC could be a key metabolic regulator in exhausted T cell metabolism and suggested that 5-ALA/SFC might synergize with anti-PD-1/PD-L1 therapy to boost the intratumoral efficacy of tumor-specific T cells. Our study not only revealed a new aspect of immune metabolism, but also paved the way to develop a strategy for combined anti-PD-1/PD-L1 cancer immunotherapy.


Assuntos
Ácido Aminolevulínico/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Ácido Cítrico/farmacologia , Compostos Ferrosos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Heme Oxigenase-1/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Antígeno Ki-67/metabolismo , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Sirtuína 1/metabolismo
10.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947138

RESUMO

Ovarian clear cell carcinoma (OCCC) is a rare but chemorefractory tumor. About 50% of all OCCC patients have inactivating mutations of ARID1A, a member of the SWI/SNF chromatin-remodeling complex. Members of the SWI/SNF remodeling have emerged as regulators of the energetic metabolism of mammalian cells; however, the role of ARID1A as a modulator of the mitochondrial metabolism in OCCCs is yet to be defined. Here, we show that ARID1A loss results in increased mitochondrial metabolism and renders ARID1A-mutated cells increasingly and selectively dependent on it. The increase in mitochondrial activity following ARID1A loss is associated with increase in c-Myc expression and increased mitochondrial number and reduction of their size consistent with a higher mitochondrial cristae/outer membrane ratio. Significantly, preclinical testing of the complex I mitochondrial inhibitor IACS-010759 showed it extends overall survival in a preclinical model of ARID1A-mutated OCCC. These findings provide for the targeting mitochondrial activity in ARID1A-mutated OCCCs.


Assuntos
Adenocarcinoma de Células Claras/tratamento farmacológico , Antineoplásicos/uso terapêutico , Proteínas de Ligação a DNA/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Oxidiazóis/uso terapêutico , Piperidinas/uso terapêutico , Fatores de Transcrição/antagonistas & inibidores , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma de Células Claras/patologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Nus , Mitocôndrias/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Oxidiazóis/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Piperidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Distribuição Aleatória , Esferoides Celulares , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Biochem Biophys Res Commun ; 558: 44-50, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33895550

RESUMO

Sickness symptoms exerted via inflammatory responses occur in several infectious and chronic diseases. A growing body of evidence suggests that altered nutrient availability and metabolism are tightly coupled to inflammatory processes. However, the relationship between metabolic shifts and the development of the sickness response has not been explored fully. Therefore, we aimed to evaluate metabolic phenotypes with a mouse model showing sickness symptoms via systemic administration of lipopolysaccharide (LPS) in the present study. LPS injection elevated the lipid utilization and circulating levels of fatty acids. It also increased the levels of ß-hydroxybutyric acid, a ketone body produced from fatty acids. We confirmed the functional connectivity between nutrient utilization and inflammatory responses and demonstrated enhanced lipid utilization in the hypothalamus providing insights into hypothalamic control of sickness responses. Collectively, these findings could help develop new therapeutic strategies to treat patients with severe sickness symptoms associated with infectious and chronic human diseases.


Assuntos
Comportamento de Doença/efeitos dos fármacos , Comportamento de Doença/fisiologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Animais , Anorexia/etiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Ácidos Graxos/sangue , Ácidos Graxos/metabolismo , Febre/etiologia , Humanos , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Consumo de Oxigênio/efeitos dos fármacos
12.
Biochem Biophys Res Commun ; 558: 57-63, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33895552

RESUMO

Nonalcoholic fatty liver disease (NAFLD) refers to a series of diseases, including simple steatosis, caused by the excessive accumulation of fat in hepatocytes, nonalcoholic steatohepatitis with inflammation and fibrosis, and more advanced forms of cirrhosis. The pathogenic mechanisms underlying fatty liver and the progression from simple fatty liver to hepatitis and cirrhosis remain unclear. One potentially unifying mechanism may be a dysregulation of free fatty acid oxidation. The oversupply of fatty acids to the liver can result in mitochondrial dysfunction leading to the accumulation of lipids in the liver. Interestingly, there have been several reports showing that inhibitors of phosphodiesterase 5 (PDE5) can increase mitochondrial biogenesis, preserve mitochondrial function in vitro. And, we have recently demonstrated that the phosphodiesterase type 5 inhibitor udenafil improves insulin sensitivity by increasing mitochondrial function in adipocytes. In this study, we aimed to examine the effects of the PDE5 inhibitor udenafil on NAFLD in the ob/ob mouse model. Treatment of ob/ob mice for 6 weeks with udenafil reduced fat mass and fasting glucose. Importantly, udenafil caused a reduction in lipid accumulation in the liver of these mice, including hepatic triglyceride (TG) and cholesterol levels. Mechanistically, udenafil decreased the proinflammatory cytokines in the liver. Also, udenafil increased the levels in the liver of the important lipolytic enzymes and the levels of several mitochondrial ß-oxidation related genes. Similar effects were seen in udenafil treated primary hepatocytes. We believe that our study makes a significant contribution to the literature because the results from our study suggest that udenafil may be an effective treatment for NAFLD by improving mitochondrial function.


Assuntos
Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Inibidores da Fosfodiesterase 5/farmacologia , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Animais , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Mediadores da Inflamação/metabolismo , Resistência à Insulina , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipólise/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos
13.
J Nutr Biochem ; 94: 108751, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33915261

RESUMO

Diets rich in mono or polyunsaturated fats have been associated with a healthy phenotype, but there is controversial evidence about coconut oil (CO), which is rich in saturated medium-chain fatty acids. Therefore, the purpose of the present work was to study whether different types of oils rich in polyunsaturated (soybean oil, SO), monounsaturated (olive oil, OO), or saturated fatty acids (coconut oil, CO) can regulate the gut microbiota, insulin sensitivity, inflammation, mitochondrial function in wild type and PPARα KO mice. The group that received SO showed the highest microbial diversity, increase in Akkermansia muciniphila, high insulin sensitivity and low grade inflammation, The OO group showed similar insulin sensitivity and insulin signaling than SO, increase in Bifidobacterium, increase in fatty acid oxidation and low grade inflammation. The CO consumption led to the lowest bacterial diversity, a 9-fold increase in the LPS concentration leading to metabolic endotoxemia, hepatic steatosis, increased lipogenesis, highest LDL-cholesterol concentration and the lowest respiratory capacity and fatty acid oxidation in the mitochondria. The absence of PPARα decreased alpha diversity and increased LPS concentration particularly in the CO group, and increased insulin sensitivity in the groups fed SO or OO. These results indicate that consuming mono or polyunsaturated fatty acids produced health benefits at the recommended intake but a high concentration of oils (three times the recommended oil intake in rodents) significantly decreased the microbial alpha-diversity independent of the type of oil.


Assuntos
Óleo de Coco/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Azeite de Oliva/farmacologia , PPAR alfa/metabolismo , Óleo de Soja/farmacologia , Animais , Bactérias/classificação , Bactérias/genética , Células Cultivadas , Biologia Computacional , DNA Bacteriano/genética , Fezes/química , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Intolerância à Glucose , Hepatócitos/efeitos dos fármacos , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , PPAR alfa/genética , RNA Bacteriano/genética , RNA Ribossômico 16S , Distribuição Aleatória , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
14.
Biomed Pharmacother ; 139: 111593, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33865018

RESUMO

BACKGROUND: Cerebral ischemic events, comprising of excitotoxicity, reactive oxygen production, and inflammation, adversely impact the metabolic-redox circuit in highly active neuronal metabolic profile which maintains energy-dependent brain activities. Therefore, we investigated neuro-regenerative potential of melatonin (Mel), a natural biomaterial secreted by pineal gland. METHODS: We specifically determined whether Mel could influence tunneling nanotubes (TNTs)-mediated transfer of functional mitochondria (Mito) which in turn may alter membrane potential, oxidative stress and apoptotic factors. In vitro studies assessed the effects of Mito on levels of cytochrome C, mitochondrial transfer, reactive oxygen species, membrane potential and mass, which were all further enhanced by Mel pre-treatment, whereas in vivo studies examined brain infarct area (BIA), neurological function, inflammation, brain edema and integrity of neurons and myelin sheath in control, ischemia stroke (IS), IS + Mito and IS + Mel-Mito group rats. RESULTS: Results showed that Mel pre-treatment significantly increased mitochondrial transfer and antioxidants, and inhibited apoptosis. Mel-pretreated Mito also significantly reduced BIA with improved neurological function. Apoptotic, oxidative-stress, autophagic, mitochondrial/DNA-damaged biomarkers indices were also improved. CONCLUSION: Conclusively, Mel is a potent biomaterial which could potentially impart neurogenesis through repairing impaired metabolic-redox circuit via enhanced TNT-mediated mitochondrial transfer, anti-oxidation, and anti-apoptotic activities in ischemia.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Melatonina/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Apoptose/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Peróxido de Hidrogênio/farmacologia , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Nanotubos , Neurogênese/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos Sprague-Dawley , Regulação para Cima
15.
Nat Commun ; 12(1): 2074, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824316

RESUMO

Thiazoline-related innate fear-eliciting compounds (tFOs) orchestrate hypothermia, hypometabolism, and anti-hypoxia, which enable survival in lethal hypoxic conditions. Here, we show that most of these effects are severely attenuated in transient receptor potential ankyrin 1 (Trpa1) knockout mice. TFO-induced hypothermia involves the Trpa1-mediated trigeminal/vagal pathways and non-Trpa1 olfactory pathway. TFOs activate Trpa1-positive sensory pathways projecting from trigeminal and vagal ganglia to the spinal trigeminal nucleus (Sp5) and nucleus of the solitary tract (NTS), and their artificial activation induces hypothermia. TFO presentation activates the NTS-Parabrachial nucleus pathway to induce hypothermia and hypometabolism; this activation was suppressed in Trpa1 knockout mice. TRPA1 activation is insufficient to trigger tFO-mediated anti-hypoxic effects; Sp5/NTS activation is also necessary. Accordingly, we find a novel molecule that enables mice to survive in a lethal hypoxic condition ten times longer than known tFOs. Combinations of appropriate tFOs and TRPA1 command intrinsic physiological responses relevant to survival fate.


Assuntos
Medo/fisiologia , Hipotermia/metabolismo , Hipóxia/metabolismo , Canal de Cátion TRPA1/metabolismo , Tiazóis/farmacologia , Animais , Bradicardia/patologia , Medo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Hipotermia/complicações , Hipóxia/complicações , Masculino , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Sensação/efeitos dos fármacos , Tiazóis/química , Fatores de Tempo , Gânglio Trigeminal/efeitos dos fármacos , Gânglio Trigeminal/metabolismo , Nervo Vago/efeitos dos fármacos
16.
Nat Commun ; 12(1): 2148, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846320

RESUMO

Deregulation of chromatin modifiers plays an essential role in the pathogenesis of medulloblastoma, the most common paediatric malignant brain tumour. Here, we identify a BMI1-dependent sensitivity to deregulation of inositol metabolism in a proportion of medulloblastoma. We demonstrate mTOR pathway activation and metabolic adaptation specifically in medulloblastoma of the molecular subgroup G4 characterised by a BMI1High;CHD7Low signature and show this can be counteracted by IP6 treatment. Finally, we demonstrate that IP6 synergises with cisplatin to enhance its cytotoxicity in vitro and extends survival in a pre-clinical BMI1High;CHD7Low xenograft model.


Assuntos
Adaptação Fisiológica , Neoplasias Cerebelares/genética , Epigênese Genética , Inositol/farmacologia , Meduloblastoma/genética , Adaptação Fisiológica/efeitos dos fármacos , Animais , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Proteínas de Ligação a DNA/metabolismo , Sinergismo Farmacológico , Epigênese Genética/efeitos dos fármacos , Humanos , Camundongos , Células-Tronco Neurais/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Fosfatidilinositóis/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas com Domínio T , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Molecules ; 26(8)2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921198

RESUMO

Cyclooxygenase (COX) and lipoxygenase (LOX) are key targets for the development of new anti-inflammatory agents. LOX, which is involved in the biosynthesis of mediators in inflammation and allergic reactions, was selected for a biochemical screening campaign to identify LOX inhibitors by employing the main natural product library of Brazilian biodiversity. Two prenyl chalcones were identified as potent inhibitors of LOX-1 in the screening. The most active compound, (E)-2-O-farnesyl chalcone, decreased the rate of oxygen consumption to an extent similar to that of the positive control, nordihydroguaiaretic acid. Additionally, studies on the mechanism of the action indicated that (E)-2-O-farnesyl chalcone is a competitive LOX-1 inhibitor. Molecular modeling studies indicated the importance of the prenyl moieties for the binding of the inhibitors to the LOX binding site, which is related to their pharmacological properties.


Assuntos
Chalconas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Inibidores de Lipoxigenase/farmacologia , Modelos Moleculares , Prenilação , Chalconas/química , Concentração Inibidora 50 , Lipoxigenase/metabolismo , Inibidores de Lipoxigenase/química , Simulação de Acoplamento Molecular , Consumo de Oxigênio/efeitos dos fármacos
18.
Goiânia; SES-GO; 12 mar. 2021. 1-5 p. fig.
Não convencional em Português | LILACS, Coleciona SUS, CONASS, SES-GO | ID: biblio-1247400

RESUMO

A pandemia do novo coronavírus trouxe grande sobrecarga aos sistemas de saúde de todo o mundo, especialmente aos países de baixa e média renda (BONG CL et al, 2020). O atraso da vacinação tem agravado a situação nestes países, aumentando o índice de novos casos e a mortalidade pela doença (WHO, 2021). Um dos problemas enfrentados tem sido a escassez da oferta de oxigênio (O2) hospitalar. Agências internacionais como Wellcome Trust, Unitaid e MS criaram uma força tarefa em busca de uma resposta emergencial para a situação, estimando a necessidade de um aporte financeiro de cerca de 90 milhões de dólares, a fim de se evitar mortes preveníveis pela falta de oxigênio para o manejo clínico dos pacientes hospitalizados por COVID-19 (USHER AD, 2021).


The pandemic of the new coronavirus has brought great burden to health systems around the world, especially to low- and middle-income countries (BONG CL et al, 2020). The delay of vaccination has aggravated the situation in these countries, increasing the rate of new cases and mortality from the disease (WHO, 2021). One of the problems faced has been the scarcity of hospital oxygen (O2) supply. International agencies such as Wellcome Trust, Unitaid and MS have set up a task force in search of an emergency response to the situation, estimating the need for a financial contribution of about US$90 million in order to avoid preventable deaths from the lack of oxygen for the clinical management of patients hospitalized by COVID-19 (USHER AD, 2021).


Assuntos
Humanos , Masculino , Feminino , Gravidez , Recém-Nascido , Lactente , Pré-Escolar , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Adulto Jovem , Oxigênio/fisiologia , Oxigênio/provisão & distribuição , Consumo de Oxigênio/efeitos dos fármacos
19.
Toxicology ; 455: 152766, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33775737

RESUMO

Azure A (AA) is a cationic molecule of the class of phenothiazines that has been applied in vitro as a photosensitising agent in photodynamic antimicrobial chemotherapy. It is a di-demethylated analogue of methylene blue (MB), which has been demonstrated to be intrinsically and photodynamically highly active on mitochondrial bioenergetics. However, as far as we know, there are no studies about the photodynamic effects of AA on mammalian mitochondria. Therefore, this investigation aimed to characterise the intrinsic and photodynamic acute effects of AA (0.540 µM) on isolated rat liver mitochondria, isolated hepatocytes, and isolated perfused rat liver. The effects of AA were assessed by evaluating several parameters of mitochondrial bioenergetics, oxidative stress, cell viability, and hepatic energy metabolism. The photodynamic effects of AA were assessed under simulated hypoxic conditions, a suitable way for mimicking the microenvironment of hypoxic solid tumour cells. AA interacted with the mitochondria and, upon photostimulation (10 min of light exposure), produced toxic amounts of reactive oxygen species (ROS), which damaged the organelle, as demonstrated by the high levels of lipid peroxidation and protein carbonylation. The photostimulated AA also depleted the GSH pool, which could compromise the mitochondrial antioxidant defence. Bioenergetically, AA photoinactivated the complexes I, II, and IV of the mitochondrial respiratory chain and the F1FO-ATP synthase complex, sharply inhibiting the oxidative phosphorylation. Upon photostimulation (10 min of light exposure), AA reduced the efficiency of mitochondrial energy transduction and oxidatively damaged lipids in isolated hepatocytes but did not decrease the viability of cells. Despite the useful photobiological properties, AA presented noticeable dark toxicity on mitochondrial bioenergetics, functioning predominantly as an uncoupler of oxidative phosphorylation. This harmful effect of AA was evidenced in isolated hepatocytes, in which AA diminished the cellular ATP content. In this case, the cells exhibited signs of cell viability reduction in the presence of high AA concentrations, but only after a long time of incubation (at least 90 min). The impairments on mitochondrial bioenergetics were also clearly manifested in intact perfused rat liver, in which AA diminished the cellular ATP content and stimulated the oxygen uptake. Consequently, gluconeogenesis and ureogenesis were strongly inhibited, whereas glycogenolysis and glycolysis were stimulated. AA also promoted the release of cytosolic and mitochondrial enzymes into the perfusate concomitantly with inhibition of oxygen consumption. In general, the intrinsic and photodynamic effects of AA were similar to those of MB, but AA caused some distinct effects such as the photoinactivation of the complex IV of the mitochondrial respiratory chain and a diminution of the ATP levels in the liver. It is evident that AA has the potential to be used in mitochondria-targeted photodynamic therapy, even under low oxygen concentrations. However, the fact that AA directly disrupts mitochondrial bioenergetics and affects several hepatic pathways that are linked to ATP metabolism, along with its ability to perturb cellular membranes and its little potential to reduce cell viability, could result in significant adverse effects especially in long-term treatments.


Assuntos
Corantes Azur/toxicidade , Metabolismo Energético/efeitos dos fármacos , Fígado/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/patologia , Masculino , Mitocôndrias Hepáticas/patologia , Consumo de Oxigênio/efeitos dos fármacos , Carbonilação Proteica/efeitos dos fármacos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo
20.
J Cereb Blood Flow Metab ; 41(4): 841-856, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33736512

RESUMO

Understanding cellular contributions to hemodynamic activity is essential for interpreting blood-based brain mapping signals. Optogenetic studies examining cell-specific influences on local hemodynamics have reported that excitatory activity results in cerebral perfusion and blood volume increase, while inhibitory activity contributes to both vasodilation and vasoconstriction. How specific subpopulations of interneurons regulate the brain's blood supply is less examined. Parvalbumin interneurons are the largest subpopulation of GABAergic neurons in the brain, critical for brain development, plasticity, and long-distance excitatory neurotransmission. Despite their essential role in brain function, the contribution of parvalbumin neurons to neurovascular coupling has been relatively unexamined. Using optical intrinsic signal imaging and laser speckle contrast imaging, we photostimulated awake and anesthetized transgenic mice expressing channelrhodopsin under a parvalbumin promoter. Increased parvalbumin activity reduced local oxygenation, cerebral blood volume, and cerebral blood flow. These "negative" hemodynamic responses were consistent within and across mice and reproducible across a broad range of photostimulus parameters. However, the sign and magnitude of the hemodynamic response resulting from increased parvalbumin activity depended on the type and level of anesthesia used. Opposed hemodynamic responses following increased excitation or parvalbumin-based inhibition suggest unique contributions from different cell populations to neurovascular coupling.


Assuntos
Circulação Cerebrovascular/fisiologia , Hemodinâmica , Parvalbuminas , Animais , Volume Sanguíneo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Circulação Cerebrovascular/efeitos dos fármacos , Channelrhodopsins/genética , Interneurônios/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neuroimagem , Consumo de Oxigênio/efeitos dos fármacos , Estimulação Luminosa , Transmissão Sináptica , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Ácido gama-Aminobutírico/fisiologia
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