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1.
Medicine (Baltimore) ; 99(34): e21821, 2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32846824

RESUMO

BACKGROUND: Traditional Chinese medicine Tongxinluo (TXL) has been widely used to treat coronary artery disease in China, since it could reduce myocardial infarct size and ischemia/reperfusion injury in both non-diabetic and diabetic conditions. It has been shown that TXL could regulate peroxisome proliferator activated receptor-α (PPAR-α), a positive modulator of angiopoietin-like 4 (Angptl4), in diabetic rats. Endothelial junction substructure components, such as VE-cadherin, are involved in the protection of reperfusion injury. Thus, we hypothesized cell-intrinsic and endothelial-specific Angptl4 mediated the protection of TXL on endothelial barrier under high glucose condition against ischemia/reperfusion-injury via PPAR-α pathway. METHODS: Incubated with high glucose medium, the human cardiac microvascular endothelial cells (HCMECs) were then exposed to oxygen-glucose-serum deprivation (2 hours) and restoration (2 hours) stimulation, with or without TXL, insulin, or rhAngptl4 pretreatment. RESULTS: TXL, insulin, and rhAngptl4 had similar protective effects on the endothelial barrier. TXL treatment reversed the endothelial barrier breakdown in HCMECs significantly as identified by decreasing endothelial permeability, upregulating the expression of JAM-A, VE-cadherin, and integrin-α5 and increasing the membrane location of VE-cadherin and integrin-α5, and these effects of TXL were as effective as insulin and rhAngptl4. However, Angptl4 knock-down with small interfering RNA (siRNA) interference and PPAR-α inhibitor MK886 partially abrogated these beneficial effects of TXL. Western blotting also revealed that similar with insulin, TXL upregulated the expression of Angptl4 in HCMECs, which could be inhibited by Angptl4 siRNA or MK886 exposure. TXL treatment increased PPAR-α activity, which could be diminished by MK886 but not by Angptl4 siRNA. CONCLUSION: These data suggest cell-intrinsic and endothelial-specific Angptl4 mediates the protection of TXL against endothelial barrier breakdown during oxygen-glucose-serum deprivation and restoration under high glucose condition partly via the PPAR-α/Angptl4 pathway.


Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Células Endoteliais/efeitos dos fármacos , Endotélio/efeitos dos fármacos , Endotélio/fisiopatologia , PPAR alfa/metabolismo , Proteína 4 Semelhante a Angiopoietina/genética , Proteína 4 Semelhante a Angiopoietina/farmacologia , Caderinas/metabolismo , Permeabilidade Capilar , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Vasos Coronários/citologia , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Glucose/farmacologia , Humanos , Indóis/farmacologia , Insulina/farmacologia , Integrina alfa5/metabolismo , Inibidores de Lipoxigenase/farmacologia , Microvasos/citologia , Oxigênio/metabolismo , Oxigênio/farmacologia , Receptores de Superfície Celular/metabolismo , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais
2.
J Med Life ; 13(2): 241-248, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32742521

RESUMO

Cell culture is one of the most commonly used techniques in the production of biological products. Many physical and chemical parameters may affect cell growth and proliferation. This study was conducted to investigate the effect of chemical components as supplements using the experimental design method, which aimed at reducing the number of experiments. For this purpose, supplements including chemical components using four levels, with three replications in suspension and batch culture conditions, were examined for 72 hours using the Taguchi experimental design method. From the experiments, it was concluded that the culture media composition had a significant impact on final cell count and pH. High concentrations of different media composition alone were insufficient to ensure higher cell count. According to the results, this insufficiency was associated with an increase of 20% in the number of final cells. In the majority of cultures, the number of final cells at 48 hours increased relative to the number of final cells at 24 hours after culturing the cells.


Assuntos
Técnicas de Cultura de Células/métodos , Vírus da Febre Aftosa/imunologia , Rim/citologia , Vacinas Virais/imunologia , Aminoácidos/farmacologia , Animais , Contagem de Células , Células Cultivadas , Cricetinae , Vírus da Febre Aftosa/efeitos dos fármacos , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Polietilenoglicóis/química , Proteínas/farmacologia , Vitaminas/farmacologia
3.
PLoS One ; 15(8): e0236822, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764772

RESUMO

Various marine fungi have been shown to produce interesting, bioactive compounds, but scaling up the production of these compounds can be challenging, particularly because little is generally known about how the producing organisms grow. Here we assessed the suitability of using 100-well BioScreen plates or 96-well plates incubated in a robot hotel to cultivate eight filamentous marine fungi, six sporulating and two non-sporulating, to obtain data on growth and substrate (glucose, xylose, galactose or glycerol) utilisation in a high throughput manner. All eight fungi grew in both cultivation systems, but growth was more variable and with more noise in the data in the Cytomat plate hotel than in the BioScreen. Specific growth rates between 0.01 (no added substrate) and 0.07 h-1 were measured for strains growing in the BioScreen and between 0.01 and 0.27 h-1 for strains in the plate hotel. Three strains, Dendryphiella salina LF304, Penicillium chrysogenum KF657 and Penicillium pinophilum LF458, consistently had higher specific growth rates on glucose and xylose in the plate hotel than in the BioScreen, but otherwise results were similar in the two systems. However, because of the noise in data from the plate hotel, the data obtained from it could only be used to distinguish between substrates which did or did not support growth, whereas data from BioScreen also provided information on substrate preference. Glucose was the preferred substrate for all strains, followed by xylose and galactose. Five strains also grew on glycerol. Therefore it was important to minimise the amount of glycerol introduced with the inoculum to avoid misinterpreting the results for growth on poor substrates. We concluded that both systems could provide physiological data with filamentous fungi, provided sufficient replicates are included in the measurements.


Assuntos
Ascomicetos/crescimento & desenvolvimento , Penicillium/crescimento & desenvolvimento , Água do Mar/microbiologia , Ascomicetos/efeitos dos fármacos , Ascomicetos/isolamento & purificação , Meios de Cultura/química , Meios de Cultura/farmacologia , Glucose/farmacologia , Glicerol/farmacologia , Penicillium/efeitos dos fármacos , Penicillium/isolamento & purificação , Xilose/farmacologia
4.
PLoS Biol ; 18(8): e3000757, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32833957

RESUMO

In eukaryotes, conserved mechanisms ensure that cell growth is coordinated with nutrient availability. Overactive growth during nutrient limitation ("nutrient-growth dysregulation") can lead to rapid cell death. Here, we demonstrate that cells can adapt to nutrient-growth dysregulation by evolving major metabolic defects. Specifically, when yeast lysine-auxotrophic mutant lys- encountered lysine limitation, an evolutionarily novel stress, cells suffered nutrient-growth dysregulation. A subpopulation repeatedly evolved to lose the ability to synthesize organosulfurs (lys-orgS-). Organosulfurs, mainly reduced glutathione (GSH) and GSH conjugates, were released by lys- cells during lysine limitation when growth was dysregulated, but not during glucose limitation when growth was regulated. Limiting organosulfurs conferred a frequency-dependent fitness advantage to lys-orgS- by eliciting a proper slow growth program, including autophagy. Thus, nutrient-growth dysregulation is associated with rapid organosulfur release, which enables the selection of organosulfur auxotrophy to better tune cell growth to the metabolic environment. We speculate that evolutionarily novel stresses can trigger atypical release of certain metabolites, setting the stage for the evolution of new ecological interactions.


Assuntos
Adaptação Fisiológica/genética , Lisina/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Nutrientes/farmacologia , Saccharomyces cerevisiae/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Evolução Biológica , Glucose/metabolismo , Glucose/farmacologia , Lisina/deficiência , Redes e Vias Metabólicas/genética , Nitrogênio/metabolismo , Nitrogênio/farmacologia , Nutrientes/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sirolimo/farmacologia , Estresse Fisiológico
5.
Ecotoxicol Environ Saf ; 204: 111005, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32738624

RESUMO

Freezing temperatures is an important stressor in the arctic regions and has a significant influence on the population dynamics and geographic distribution of terrestrial invertebrates. Toxic metals in the environment can interfere with protective cold-acclimation responses of organisms. It is therefore important to evaluate the combined effects of cold stress and environmental contaminants. Here, we aimed to investigate the effects of Hg (HgCl2) on various physiological aspects of freeze-tolerance in the earthworm (Enchytraeus albidus). We measured the levels of the cryoprotectant glucose, the glycogen content (source of glucose molecules for cryoprotection and fuel for metabolism), and changes in the composition of membrane phospholipid fatty acids (PLFA) as an indicator of lipid peroxidation. Freezing at -6 °C had no effect on survival in uncontaminated soil, however, survival of freezing in Hg contaminated soil was clearly reduced, especially at extended exposure times. Thus, the LC50 value in frozen soil decreased from 8.3 mg Hg kg-1 (when exposed for 17 days) to only 4.2 mg Hg kg-1 after 36 days' exposure indicating that combined effects of Hg and freezing became larger at prolonged exposure times. Hg caused a depletion of glycogen reserves (almost 50% at 12 mg kg-1 dry soil), but despite this effect worms were able to maintain a constant cryoprotectant level (about 0.12 mg glucose mg-1 dry weight) at all Hg concentrations. Hg had clear negative effects on the proportion of unsaturated PLFAs, which could be an indication of lipid peroxidation. Since a high proportion of unsaturated fatty acids in the membrane is important for invertebrate freeze-tolerance, our results suggest that the negative effect of Hg on freeze-tolerance in E. albidus is related to degraded membrane functionality at low temperature.


Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Congelamento , Cloreto de Mercúrio/efeitos adversos , Oligoquetos/efeitos dos fármacos , Animais , Crioprotetores/farmacologia , Relação Dose-Resposta a Droga , Ácidos Graxos/metabolismo , Glucose/farmacologia , Glicogênio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Mercúrio/efeitos adversos
6.
PLoS Biol ; 18(6): e3000732, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32603375

RESUMO

Coordination of gene expression with nutrient availability supports proliferation and homeostasis and is shaped by protein acetylation. Yet how physiological/pathological signals link acetylation to specific gene expression programs and whether such responses are cell-type-specific is unclear. AMP-activated protein kinase (AMPK) is a key energy sensor, activated by glucose limitation to resolve nutrient supply-demand imbalances, critical for diabetes and cancer. Unexpectedly, we show here that, in gastrointestinal cancer cells, glucose activates AMPK to selectively induce EP300, but not CREB-binding protein (CBP). Consequently, EP300 is redirected away from nuclear receptors that promote differentiation towards ß-catenin, a driver of proliferation and colorectal tumorigenesis. Importantly, blocking glycogen synthesis permits reactive oxygen species (ROS) accumulation and AMPK activation in response to glucose in previously nonresponsive cells. Notably, glycogen content and activity of the ROS/AMPK/EP300/ß-catenin axis are opposite in healthy versus tumor sections. Glycogen content reduction from healthy to tumor tissue may explain AMPK switching from tumor suppressor to activator during tumor evolution.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Neoplasias Colorretais/metabolismo , Proteína p300 Associada a E1A/metabolismo , Glucose/farmacologia , Animais , Proteína de Ligação a CREB/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Ativação Enzimática/efeitos dos fármacos , Glicogênio/metabolismo , Camundongos Endogâmicos C57BL , Ligação Proteica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , beta Catenina/metabolismo
7.
DNA Cell Biol ; 39(9): 1700-1710, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32721233

RESUMO

The increased secretion of glucagon-like peptide-1 (GLP-1) after Roux-en-Y gastric bypass (RYGB) is regarded as the main reason for the improvement of blood glucose. However, the single-nucleotide polymorphisms (SNPs) of GLP-1 Receptor (GLP1R) impair receptor function, subsequently affecting ß cell insulin secretion function, ultimately affecting the efficacy of RYGB. In this study, we revealed that two SNPs in GLP1R gene, rs3765467 and rs10305492, could significantly reduce the insulin secreted by ß cells and the cyclic AMP concentration, whereas promote ß cell apoptosis. Under high glucose exposure, rs3765467 and rs10305492 impaired ß cell secretion of insulin and ß cell viability in the same way; in other words, GLP1R rs3765467 and rs10305492 exert an effect on pancreatic ß cell glucose-stimulated insulin secretion. Moreover, GLP-1 antagonist Exendin (9-39) further enhanced, whereas GLP-1 agonist Exendin-4 partially attenuated the effects of SNPs on the functions and apoptosis of ß cells. In conclusion, the rs3765467 and rs10305492 SNPs in GLP1R show to exert a critical effect on regulating insulin secretory capacity of ß cells and ß cell mass. Through leading to the dysfunction and apoptosis of ß cells, GLP1R rs3765467 and rs10305492 might also impair GLP-1 interaction with GLP1R, therefore attenuating the therapeutic effect of RYGB.


Assuntos
Apoptose , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Polimorfismo de Nucleotídeo Único , Animais , Linhagem Celular , Células Cultivadas , Exenatida/farmacologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Mutação , Ratos
8.
PLoS Biol ; 18(6): e3000741, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32520929

RESUMO

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.


Assuntos
Metabolômica , Mitocôndrias/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo , Trifosfato de Adenosina/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Elétrons , Glucose/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oxirredução , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transcriptoma/genética , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/genética
9.
Obesity (Silver Spring) ; 28(7): 1270-1282, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32538511

RESUMO

OBJECTIVE: This study aimed to investigate whether the NLRP3 inflammasome in Kupffer cells (KCs) can be activated in response to high glucose (HG) and to evaluate its influence on hepatic insulin sensitivity. METHODS: Primary KCs and hepatocytes were isolated from mice, and lipid accumulation, glucose output, and insulin sensitivity of hepatocytes were investigated after culturing either alone or with KCs exposed to HG. The influence of HG-induced NLRP3 inflammasome activation in KCs on insulin sensitivity of hepatocytes was examined. Treatment with gadolinium trichloride caused KC depletion, and, subsequently, a streptozotocin-induced hyperglycemic mouse model was used to confirm the influence of KCs on hepatic insulin sensitivity. RESULTS: Hepatocytes cocultured with KCs showed enhanced lipid accumulation, glucose output, and impaired insulin sensitivity when exposed to HG. Enhanced NLRP3 inflammasome activation was also evident in both hepatocytes and KCs. Moreover, KCs that were pretreated with caspase-1 inhibitor, NLRP3 inhibitor, and NLRP3 small interfering RNA corrected coculture-induced aberrances in insulin action and NLRP3 inflammasome activation in hepatocytes. KC coculture also increased interleukin-1ß (IL-1ß)-mediated nuclear factor-κB (NF-κB) activation in hepatocytes. In hyperglycemic mice, KC depletion inhibited NLRP3 inflammasome activation and improved hepatic insulin sensitivity. CONCLUSIONS: NLRP3 inflammasome activation impaired insulin sensitivity through KC-derived IL-1ß-mediated NF-κB activation in hepatocytes exposed to HG.


Assuntos
Glucose/farmacologia , Inflamassomos/fisiologia , Resistência à Insulina , Macrófagos do Fígado/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Resistência à Insulina/genética , Macrófagos do Fígado/efeitos dos fármacos , Macrófagos do Fígado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética
10.
PLoS One ; 15(6): e0235118, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579601

RESUMO

During diabetes, renal proximal tubular cells (PTC) are exposed to a combination of high glucose and hypoxic conditions, which plays a relevant role in the development of diabetic kidney disease (DKD). In this work, a time-series proteomic study was performed to analyse the effect of a diabetic-like microenvironment induced changes on HK-2 cells, a human cell line derived from normal proximal tubular epithelial cells. Cells simultaneously exposed to high glucose (25 mM) and hypoxia (1% O2) were compared to cells in control conditions for up to 48 h. Diabetic conditions increased the percentage of death cells after 24 and 48 h, but no differences in the protein/cell ratio were found. The relative protein quantification using dimethyl-labeling and UHPLC-MS/MS analysis allowed the identification of 317, 296 and 259 proteins at 5, 24 and 48 h, respectively. The combination of statistical and time expression profile analyses indicated an increased expression of proteins involved in glycolysis, and a decrease of cytoskeletal-related proteins. The exposure of HK-2 cells to high glucose and hypoxia reproduces some of the effects of diabetes on PTC and, with the limitations inherent to in vitro studies, propose new mechanisms and targets to be considered in the management of DKD.


Assuntos
Células Epiteliais/metabolismo , Glucose/metabolismo , Túbulos Renais Proximais/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Hipóxia Celular , Linhagem Celular , Cromatografia Líquida de Alta Pressão/métodos , Nefropatias Diabéticas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glucose/farmacologia , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Espectrometria de Massas em Tandem/métodos , Fatores de Tempo
11.
Ren Fail ; 42(1): 463-473, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32408786

RESUMO

Objective: This report was designed to assess the functional role of miR-218/dachshund family transcription factor 1 (DACH1) in diabetic kidney disease (DKD) and investigate its possible molecular mechanism.Materials and Methods: From the GEO database, we downloaded different datasets for analyzing the expression of miR-218 and DACH1 in DKD. TargetScan was adopted to predict the binding sites between miR-218 and DACH1, which was further verified by dual-luciferase reporter assays. The renal proximal tubule cells (HK-2) treated with high glucose (HG) were used as an in vitro model. QRT-PCR and western blot were used to determine the expression of DACH1 and other relative factors. Cell counting kit-8 and flow cytometer were applied to detect cell viability and apoptosis. The levels of inflammatory cytokines were determined by an ELISA assay.Results: A prominent raise of miR-218 was observed in DKD through bioinformatics analysis, which was further confirmed in the HG-induced model. DACH1 is a target of miR-218. miR-218 reduced cell viability and induced apoptosis by negatively regulating DACH1. Moreover, upregulating miR-218 in HG models increased the concentrations of pro-inflammatory cytokines TNF-α and IL-1ß, reduced the level of anti-inflammatory cytokine IL-10, and promoted the epithelial-mesenchymal transition (EMT) process, which is possibly achieved by targeting DACH1. While downregulating miR-218 showed the opposite results.Conclusion: These data demonstrated that, under an in vitro HG environment, miR-218 suppressed the HK-2 cells proliferation, promoted apoptosis, caused an inflammatory response, and facilitated the EMT process largely by targeting DACH1, providing an insight into the therapeutic intervention of DKD.


Assuntos
Apoptose/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Proteínas do Olho/metabolismo , Glucose/farmacologia , MicroRNAs/genética , Fatores de Transcrição/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas do Olho/genética , Humanos , Inflamação , Rim , Fatores de Transcrição/genética
12.
Nat Commun ; 11(1): 2127, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358544

RESUMO

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular events in humans with type 2 diabetes (T2D); however, the underlying mechanism remains unclear. Activation of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome and subsequent interleukin (IL)-1ß release induces atherosclerosis and heart failure. Here we show the effect of SGLT2 inhibitor empagliflozin on NLRP3 inflammasome activity. Patients with T2D and high cardiovascular risk receive SGLT2 inhibitor or sulfonylurea for 30 days, with NLRP3 inflammasome activation analyzed in macrophages. While the SGLT2 inhibitor's glucose-lowering capacity is similar to sulfonylurea, it shows a greater reduction in IL-1ß secretion compared to sulfonylurea accompanied by increased serum ß-hydroxybutyrate (BHB) and decreased serum insulin. Ex vivo experiments with macrophages verify the inhibitory effects of high BHB and low insulin levels on NLRP3 inflammasome activation. In conclusion, SGLT2 inhibitor attenuates NLRP3 inflammasome activation, which might help to explain its cardioprotective effects.


Assuntos
Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Idoso , Animais , Doenças Cardiovasculares/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/farmacologia , Humanos , Insulina/metabolismo , Interleucina-1beta/metabolismo , Cetonas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Pessoa de Meia-Idade , Inibidores do Transportador 2 de Sódio-Glicose , Compostos de Sulfonilureia/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
13.
Am J Physiol Regul Integr Comp Physiol ; 319(1): R69-R78, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32432916

RESUMO

The production of H2S and its effect on bioenergetics in mammalian cells may be evolutionarily preserved. Erythrocytes of birds, but not those of mammals, have a nucleus and mitochondria. In the present study, we report the endogenous production of H2S in chicken erythrocytes, which was mainly catalyzed by 3-mercaptopyruvate sulfur transferase (MST). ATP content of erythrocytes was increased by MST-generated endogenous H2S under normoxic, but not hypoxic, conditions. NaHS, a H2S salt, increased ATP content under normoxic, but not hypoxic, conditions. ATP contents in the absence or presence of NaHS were eliminated by different inhibitors for mitochondrial electron transport chain in chicken erythrocytes. Succinate and glutamine, but not glucose, increased ATP content. NaHS treatment similarly increased ATP content in the presence of glucose, glutamine, or succinate, respectively. Furthermore, the expression and activity of sulfide:quinone oxidoreductase were enhanced by NaHS. The structural integrity of chicken erythrocytes was largely maintained during 2-wk NaHS treatment in vitro, whereas most of the erythrocytes without NaHS treatment were lysed. In conclusion, H2S may regulate cellular bioenergetics as well as cell survival of chicken erythrocytes, in which the functionality of the electron transport chain is involved. H2S may have different regulatory roles and mechanisms in bioenergetics of mammalian and bird cells.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Eritrócitos/metabolismo , Sulfeto de Hidrogênio/farmacologia , Trifosfato de Adenosina/sangue , Animais , Galinhas , Transporte de Elétrons/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Feminino , Glucose/farmacologia , Glutamina/farmacologia , Hipóxia/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ácido Succínico/farmacologia , Sulfurtransferases/metabolismo
14.
Am J Physiol Regul Integr Comp Physiol ; 319(1): R96-R105, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32459971

RESUMO

The rectal gland of the spiny dogfish Squalus acanthias secretes a salt solution isosmotic with plasma that maintains the salt homeostasis of the fish. It secretes salt against an electrochemical gradient that requires the expenditure of energy. Isolated rectal glands perfused without glucose secrete salt, albeit at a rate about 30% of glands perfused with 5 mM glucose. Gradually reducing the glucose concentration is associated with a progressive decrease in the secretion of chloride. The apparent Km for the exogenous glucose-dependent chloride secretion is around 2 mM. Phloretin and cytochalasin B, agents that inhibit facilitated glucose carriers of the solute carrier 2 (Slc2) family such as glucose transporter 2 (GLUT2), do not inhibit the secretion of chloride by the perfused rectal glands. Phloridzin, which inhibits Slc5 family of glucose symporters, or α-methyl-d-glucoside, which competitively inhibits the uptake of glucose through Slc5 symporters, inhibit the secretion of chloride. Thus the movement of glucose into the rectal gland cells appears to be mediated by a sodium-glucose symporter. Sodium-glucose cotransporter 1 (SGLT1), the first member of the Slc5 family of sodium-linked glucose symporters, was cloned from the rectal gland. No evidence of GLUT2 was found. The persistence of secretion of chloride in the absence of glucose in the perfusate suggests that there is an additional source of energy within the cells. The use of 2-mercapto-acetate did not result in any change in the secretion of chloride, suggesting that the oxidation of fatty acids is not the source of energy for the secretion of chloride. Perfusion of isolated glands with KCN in the absence of glucose further reduces the secretion of chloride but does not abolish it, again suggesting that there is another source of energy within the cells. Glucose was measured in the rectal gland cells and found to be at concentrations in the range of that in the perfusate. Glycogen measurements indicated that there are significant stores of glucose in the rectal gland. Moreover, glycogen synthase was partially cloned from rectal gland cells. The open reading frame of glycogen phosphorylase was also cloned from rectal gland cells. Measurements of glycogen phosphorylase showed that the enzyme is mostly in its active form in the cells. The cells of the rectal gland of the spiny dogfish require exogenous glucose to fully support the active secretion of salt. They have the means to transport glucose into the cells in the form of SGLT1. The cells also have an endogenous supply of glucose as glycogen and have the necessary elements to synthesize, store, and hydrolyze it.


Assuntos
Cloretos/metabolismo , Glucose/metabolismo , Glândula de Sal/metabolismo , Squalus/metabolismo , Animais , Sequência de Bases , Glucose/farmacologia , Transportador de Glucose Tipo 2/metabolismo , Glicogênio/metabolismo , Glicogênio Fosforilase/metabolismo , Glicogênio Sintase/metabolismo , Homeostase , Técnicas In Vitro , Cianeto de Potássio/farmacologia , Glândula de Sal/efeitos dos fármacos , Transportador 1 de Glucose-Sódio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo II/metabolismo
15.
PLoS One ; 15(4): e0224251, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32339204

RESUMO

PURPOSE: To determine the effect of decorin on oxidative stress and apoptosis of human lens epithelial (HLE) cells under high glucose condition. METHODS: HLE cell line (HLEB3) was incubated in normal glucose (5.5 mM) or high glucose (60 mM) medium. Decorin (50 nM) was applied 2 hours before high glucose medium was added. Apoptosis detection was executed by flow cytometry and western blotting (analysis of bcl-2 and bax). Oxidative stress level was measured by the generation of reactive oxygen species (ROS), glutathione peroxidase (GSH) and superoxide dismutase (SOD). P38 mitogen-activated protein kinase (MAPK) phosphorylation, the expression of p22phox of HLE cells and human lens anterior capsules were detected by western blotting. Small interfering RNA transfection to p22phox and p38 MAPK was also carried out on HLEB3. RESULTS: High glucose caused HLE cells oxidative stress and apoptosis exhibiting the increase of apoptotic cells and ROS production and decrease of bcl-2/bax ratio, GSH/GSSG ration and SOD activity. P22phox and phospho-p38 MAPK were upregulated in high glucose treated HLEB3 cells. Knocking down p22phox or p38 by siRNAs can reduce high glucose induced cell apoptosis and oxidative stress level. Silencing p22phox by siRNA can downregulate the phosphorylation of p38 MAPK. Decorin can inhibit the apoptosis, oxidative stress level and the induction of p22phox and phospho-p38 of HLEB3 induced by high glucose. Furthermore, the expression of p22phox and p38 were found significantly increased in lens anterior capsules of diabetic cataract patients compared to that of normal age-related cataract patients. CONCLUSIONS: Results showed that p22phox-p38 pathway may be participated in high glucose induced lens epithelial cell injury, decorin may inhibit the high glucose induced apoptosis and oxidative stress injury by suppressing this pathway in part.


Assuntos
Cápsula Anterior do Cristalino/citologia , Antioxidantes/farmacologia , Apoptose , Decorina/farmacologia , Células Epiteliais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Idoso , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Glucose/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo
16.
Circ Heart Fail ; 13(4): e006409, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32264717

RESUMO

BACKGROUND: Ischemic tolerance of donor hearts has a major impact on the efficiency in utilization and clinical outcomes. Molecular events during storage may influence the severity of ischemic injury. METHODS: RNA sequencing was used to study the transcriptional profile of the human left ventricle (LV, n=4) and right ventricle (RV, n=4) after 0, 4, and 8 hours of cold storage in histidine-tryptophan-ketoglutarate preservation solution. Gene set enrichment analysis and gene ontology analysis was used to examine transcriptomic changes with cold storage. Terminal deoxynucleotidyl transferase 2´-Deoxyuridine, 5´-Triphosphate nick end labeling and p65 staining was used to examine for cell death and NFκB activation, respectively. RESULTS: The LV showed activation of genes related to inflammation and allograft rejection but downregulation of oxidative phosphorylation and fatty acid metabolism pathway genes. In contrast, inflammation-related genes were down-regulated in the RV and while oxidative phosphorylation genes were activated. These transcriptomic changes were most significant at the 8 hours with much lower differences observed between 0 and 4 hours. RNA velocity estimates corroborated the finding that immune-related genes were activated in the LV but not in the RV during storage. With increasing preservation duration, the LV showed an increase in nuclear translocation of NFκB (p65), whereas the RV showed increased cell death close to the endocardium especially at 8 hours. CONCLUSIONS: Our results demonstrated that the LV and RV of human donor hearts have distinct responses to cold ischemic storage. Transcriptomic changes related to inflammation, oxidative phosphorylation, and fatty acid metabolism pathways as well as cell death and NFκB activation were most pronounced after 8 hours of storage.


Assuntos
Temperatura Baixa/efeitos adversos , Transplante de Coração , Ventrículos do Coração/metabolismo , Preservação de Órgãos , Disfunção Primária do Enxerto/genética , Transcriptoma , Apoptose/efeitos dos fármacos , Apoptose/genética , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Perfilação da Expressão Gênica , Glucose/farmacologia , Transplante de Coração/efeitos adversos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Humanos , Inflamação/genética , Inflamação/patologia , Manitol/farmacologia , Preservação de Órgãos/efeitos adversos , Soluções para Preservação de Órgãos/farmacologia , Cloreto de Potássio/farmacologia , Disfunção Primária do Enxerto/patologia , Disfunção Primária do Enxerto/prevenção & controle , Procaína/farmacologia , Fatores de Risco , Fatores de Tempo , Transcriptoma/efeitos dos fármacos
17.
J Anim Sci ; 98(4)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32249288

RESUMO

Early lesions of osteochondrosis (OC) are exhibited by regions of cartilage retention along the growth plate and articular cartilage. Progression of OC lesions may impair locomotion and necessitate euthanasia in adherence to animal welfare guides. Little is known about the role of nutrition in the initiation and early stages of OC. However, dietary components are commonly implicated as predisposing factors. In this study, diets were altered as an attempt to induce early stage OC lesions under controlled conditions. At 8 wk of age, 96 crossbred gilts (body weight [BW] = 17.4 ± 0.18 kg) were randomly assigned to one of four corn-soybean meal-based diets (four pens per diet, six pigs per pen) to assess diet effects on the number and volume of OC lesions in the distal femur. Diets included a non-pelleted control diet (Ctl); Ctl plus 20% glucose (Glc); the Ctl with increased concentrations of lysine, Ca, and P (+CaP); and the +CaP diet in a pelleted form (PEL). Femurs were collected from pigs euthanized at either 14-wk (Wk 14) or 24-wk (Wk 14) of age for assessments of OC lesions. Based on a mixed model analysis with pen as the experimental unit, dietary treatments did not affect final BW (129.3 ± 3.8 kg) or average daily gain (ADG) (1.00 ± 0.03 kg/d) over the trial. As expected, pigs fed PEL and Glc diets were more efficient (P < 0.05) in feed conversion compared with Ctl and +CaP. Using femurs as the experimental unit at Wk 14 (collected from two of the six pigs per pen), bone mineral content, determined by dual-energy x-ray absorptiometry scans, was greater (P < 0.05) in pigs fed +CaP and PEL than Ctl or Glc diets; however, only +CaP group differed (P < 0.05) at Wk 24 (collected from four pigs per pen). Computed tomography (CT) scans of femurs were reconstructed as three-dimensional images to allow detection of the number, volume, and surface area of lesions in distal growth plates. At Wk 14, pigs fed Ctl had fewer number of lesions (P < 0.05); however, no differences were detected among dietary treatments in lesion volume or lesion surface area. Pigs had fewer lesions at Wk 24 than Wk 14; however, differences were not detected among dietary treatments. At Wk 24, pigs fed Ctl diets had the greatest lesion volume among dietary treatments (P < 0.05). In conclusion, none of the pigs exhibited symptoms of lameness regardless of dietary treatment or OC lesion traits. Diet modifications due to pelleting or inclusion of rapidly digestible ingredients, such as glucose, did not increase prevalence or size of OC lesions. Image analysis of CT scans was a reliable method to quantify the number, size, and location of OC lesions.


Assuntos
Suplementos Nutricionais/análise , Osteocondrose/veterinária , Doenças dos Suínos/epidemiologia , Absorciometria de Fóton/veterinária , Ração Animal/análise , Animais , Peso Corporal , Cálcio/farmacologia , Dieta/veterinária , Feminino , Fêmur/diagnóstico por imagem , Glucose/farmacologia , Incidência , Coxeadura Animal/diagnóstico por imagem , Coxeadura Animal/epidemiologia , Lisina/farmacologia , Osteocondrose/diagnóstico por imagem , Osteocondrose/epidemiologia , Fósforo/farmacologia , Distribuição Aleatória , Soja , Suínos , Doenças dos Suínos/diagnóstico por imagem , Tomografia Computadorizada por Raios X/veterinária , Zea mays
18.
Acta Diabetol ; 57(9): 1101-1109, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32280999

RESUMO

AIMS: CXXC finger protein 4 (CXXC4) is an identified negative regulator of the Wnt/ß-catenin pathway, and it is involved in cancer cell proliferation. In this study, we sought to clarify whether CXXC4 is involved in glucose-stimulated ß-cell proliferation. MATERIALS AND METHODS: We investigated the biological function of CXXC4 in glucose-induced ß-cell proliferation, and we investigated the underlying mechanism of this activity. First, we analyzed CXXC4 expression in established rat models treated for 24 h with a high glucose infusion and in INS-1 cells and primary rat islets treated with different concentrations of glucose. Subsequently, we used an adenovirus to overexpress CXXC4 in INS-1 cells and primary islets. The proliferation rate of ß-cells was evaluated by CCK-8 and EdU incorporation methods. Cell cycle analysis was performed by flow cytometry. Finally, the Wnt signaling pathway and its downstream genes were assessed by Western blot. RESULTS: CXXC4 mRNA levels were significantly lower in islets isolated from glucose-infused rats than they were in those isolated from saline-infused rats. Decreased expression of CXXC4 also correlated with high glucose treatment of INS-1 cells and primary rat ß-cells. Furthermore, adenovirus-mediated overexpression of CXXC4 inhibited cell proliferation induced by the high glucose treatment in vitro, which was mechanistically mediated by Wnt signaling and a decrease in cyclin D2 expression. CONCLUSIONS: Glucose inhibits CXXC4 expression and hence promotes pancreatic ß-cell proliferation. Our findings may provide a new potential target for the treatment of diabetes.


Assuntos
Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Animais , Proliferação de Células/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética
19.
Am J Physiol Lung Cell Mol Physiol ; 318(6): L1158-L1164, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32267720

RESUMO

Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease, but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus, to date, our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture, or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic, but not mitochondrial, ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium, and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes.


Assuntos
Ar , Diferenciação Celular , Sistemas Computacionais , Metabolismo Energético , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Nariz/citologia , Ácidos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Glucose/farmacologia , Humanos , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/metabolismo , Metabolômica
20.
PLoS One ; 15(3): e0224344, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32176701

RESUMO

A key event in the development of both major forms of diabetes is the loss of functional pancreatic islet ß-cell mass. Strategies aimed at enhancing ß-cell regeneration have long been pursued, but methods for reliably inducing human ß-cell proliferation with full retention of key functions such as glucose-stimulated insulin secretion (GSIS) are still very limited. We have previously reported that overexpression of the homeobox transcription factor NKX6.1 stimulates ß-cell proliferation, while also enhancing GSIS and providing protection against ß-cell cytotoxicity through induction of the VGF prohormone. We developed an NKX6.1 pathway screen by stably transfecting 832/13 rat insulinoma cells with a VGF promoter-luciferase reporter construct, using the resultant cell line to screen a 630,000 compound chemical library. We isolated three compounds with consistent effects to stimulate human islet cell proliferation, but not expression of NKX6.1 or VGF, suggesting an alternative mechanism of action. Further studies of the most potent of these compounds, GNF-9228, revealed that it selectively activates human ß-cell relative to α-cell proliferation and has no effect on δ-cell replication. In addition, pre-treatment, but not short term exposure of human islets to GNF-9228 enhances GSIS. GNF-9228 also protects 832/13 insulinoma cells against ER stress- and inflammatory cytokine-induced cytotoxicity. GNF-9228 stimulates proliferation via a mechanism distinct from recently emergent DYRK1A inhibitors, as it is unaffected by DYRK1A overexpression and does not activate NFAT translocation. In conclusion, we have identified a small molecule with pleiotropic positive effects on islet biology, including stimulation of human ß-cell proliferation and insulin secretion, and protection against multiple agents of cytotoxic stress.


Assuntos
Proliferação de Células/efeitos dos fármacos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular Tumoral , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/patologia , Glucose/farmacologia , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/patologia , Insulinoma/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Ratos
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