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1.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360935

RESUMO

Proximal tubules (PTs) take up most of the glucose in the glomerular filtrate and return it to peritubular capillary blood. Sodium-glucose cotransporter 2 (SGLT2) at the apical membrane takes up glucose into the cell. Glucose then flows across the cells and is transported to the interstitium via glucose transporter 2 (GLUT2) at the basolateral membrane. However, glucose transport under SGLT2 inhibition remains poorly understood. In this study, we evaluated the dynamics of a fluorescent glucose analog, 2-NBDG, in the PTs of live mice treated with or without the SGLT2 inhibitor, luseogliflozin. We employed real-time multiphoton microscopy, in which insulin enhanced 2-NBDG uptake in skeletal muscle. Influx and efflux of 2-NBDG in PT cells were compared under hypo-, normo-, and hyperglycemic conditions. Luseogliflozin did not exert significant effects on glucose influx parameters under any level of blood glucose. Our results suggest that blood glucose level per se does not alter glucose influx or efflux kinetics in PTs. In conclusion, neither SGLT2 inhibition nor blood glucose level affect glucose uptake kinetics in PTs. The former was because of glucose influx through basolateral GLUT2, which is an established bidirectional transporter.


Assuntos
Transporte Biológico/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Glucose/metabolismo , Túbulos Renais Proximais , Sorbitol/análogos & derivados , Animais , Linhagem Celular , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Sorbitol/farmacologia
2.
Int Heart J ; 62(3): 592-600, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054000

RESUMO

The clinical evidence is accumulating since 2015 that anti-diabetic sodium-glucose cotransporter 2 (SGLT2) inhibitors have the beneficial effect of cardiovascular and, recently, renal protection. Although it is not well analyzed how the transfer of this new evidence into daily practice has expedited, we hypothesize that the recent usage of the drugs is positively associated with several certified cardiologists in each region.The 2016 annual and 2016-2017 increased number of SGLT2 inhibitor tablets, based on the National Database of Health Insurance Claims and Specific Health Checkups of Japan, were divided by the estimated number of patients with type 2 diabetes mellitus for each of the 47 prefectures. Then, regression analyses were performed to investigate the potential association of the number of certified cardiologists with the drug prescription.The 2016 prescription of ipragliflozin, dapagliflozin, luseogliflozin, canagliflozin, and empagliflozin was 2.7- to 4.4-fold different between prefectures. The 2016-2017 increased prescription volume also varied among prefectures by as large as 7.3-fold for ipragliflozin. Regression analysis revealed that the annual and increased prescription volume of all the SGLT2 inhibitors except luseogliflozin were higher in regions with more certified cardiologists (P < 0.05), even after adjusting for regional parameters.In conclusion, the regional number of certified cardiologists was positively associated with a 2016 annual of and 2016-2017 increase in SGLT2 inhibitor prescription amount, implying an early adopter role of clinical experts in healthcare delivery.


Assuntos
Cardiologistas/estatística & dados numéricos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Prescrições/estatística & dados numéricos , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Compostos Benzidrílicos/farmacologia , Compostos Benzidrílicos/uso terapêutico , Canagliflozina/farmacologia , Canagliflozina/uso terapêutico , Sistema Cardiovascular/efeitos dos fármacos , Análise de Dados , Feminino , Glucosídeos/farmacologia , Glucosídeos/uso terapêutico , Humanos , Japão/epidemiologia , Rim/efeitos dos fármacos , Masculino , Análise de Regressão , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Sorbitol/análogos & derivados , Sorbitol/farmacologia , Sorbitol/uso terapêutico , Tiofenos/farmacologia , Tiofenos/uso terapêutico
3.
Nucleic Acids Res ; 49(1): 383-399, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33313903

RESUMO

Translational control is essential in response to stress. We investigated the translational programmes launched by the fission yeast Schizosaccharomyces pombe upon five environmental stresses. We also explored the contribution of defence pathways to these programmes: The Integrated Stress Response (ISR), which regulates translation initiation, and the stress-response MAPK pathway. We performed ribosome profiling of cells subjected to each stress, in wild type cells and in cells with the defence pathways inactivated. The transcription factor Fil1, a functional homologue of the yeast Gcn4 and the mammalian Atf4 proteins, was translationally upregulated and required for the response to most stresses. Moreover, many mRNAs encoding proteins required for ribosome biogenesis were translationally downregulated. Thus, several stresses trigger a universal translational response, including reduced ribosome production and a Fil1-mediated transcriptional programme. Surprisingly, ribosomes stalled on tryptophan codons upon oxidative stress, likely due to a decrease in charged tRNA-Tryptophan. Stalling caused ribosome accumulation upstream of tryptophan codons (ribosome queuing/collisions), demonstrating that stalled ribosomes affect translation elongation by other ribosomes. Consistently, tryptophan codon stalling led to reduced translation elongation and contributed to the ISR-mediated inhibition of initiation. We show that different stresses elicit common and specific translational responses, revealing a novel role in Tryptophan-tRNA availability.


Assuntos
Códon , Estresse Oxidativo/genética , Elongação Traducional da Cadeia Peptídica , RNA de Transferência de Triptofano/genética , Ribossomos/metabolismo , Schizosaccharomyces/genética , Triptofano/genética , Compostos de Cádmio/farmacologia , Fator de Iniciação 2 em Eucariotos/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Temperatura Alta , Peróxido de Hidrogênio/farmacologia , Sistema de Sinalização das MAP Quinases , Metanossulfonato de Metila/farmacologia , Proteínas Quinases Ativadas por Mitógeno/deficiência , Pressão Osmótica , RNA Fúngico/genética , RNA Mensageiro/genética , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Sorbitol/farmacologia , Sulfatos/farmacologia
4.
Molecules ; 25(24)2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33339401

RESUMO

OBJECTIVE: This study evaluated the antifungal activity of cinnamaldehyde on Candida spp. In vitro and in situ assays were carried out to test cinnamaldehyde for its anti-Candida effects, antibiofilm activity, effects on fungal micromorphology, antioxidant activity, and toxicity on keratinocytes and human erythrocytes. Statistical analysis was performed considering α = 5%. RESULTS: The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of cinnamaldehyde ranged from 18.91 µM to 37.83 µM. MIC values did not change in the presence of 0.8 M sorbitol, whereas an 8-fold increase was observed in the presence of ergosterol, suggesting that cinnamaldehyde may act on the cell membrane, which was subsequently confirmed by docking analysis. The action of cinnamaldehyde likely includes binding to enzymes involved in the formation of the cytoplasmic membrane in yeast cells. Cinnamaldehyde-treated microcultures showed impaired cellular development, with an expression of rare pseudo-hyphae and absence of chlamydoconidia. Cinnamaldehyde reduced biofilm adherence by 64.52% to 33.75% (p < 0.0001) at low concentrations (378.3-151.3 µM). Cinnamaldehyde did not show antioxidant properties. CONCLUSIONS: Cinnamaldehyde showed fungicidal activity through a mechanism of action likely related to ergosterol complexation; it was non-cytotoxic to keratinocytes and human erythrocytes and showed no antioxidant activity.


Assuntos
Acroleína/análogos & derivados , Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida/fisiologia , Acroleína/química , Acroleína/metabolismo , Acroleína/farmacologia , Antifúngicos/química , Antifúngicos/metabolismo , Antioxidantes/química , Sítios de Ligação , Candida/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ergosterol/química , Ergosterol/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Sorbitol/química , Sorbitol/farmacologia , Esqualeno Mono-Oxigenase/química , Esqualeno Mono-Oxigenase/metabolismo
5.
Arch Oral Biol ; 119: 104886, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32932149

RESUMO

OBJECTIVE: Among the preventive and therapeutic options available for dental caries, sugar alcohols (xylitol and sorbitol) have been widely promoted as oral healthcare products due to its perceived anticariogenic effect. However, the therapeutic efficacy of these sugar alcohols against Streptococcus mutans and Candida albicans in a sucrose supplemented environment, as found in disease-prone conditions in the oral cavity, has not been adequately investigated. METHODS: Single and mixed-species biofilm formation was evaluated in medium with different concentrations of xylitol, sorbitol with or without sucrose supplementation. Biofilm quantification methods such as crystal violet assay, XTT assay, CFU counting complemented with confocal and electron microscopic techniques were used. RESULTS: Under sucrose-free conditions, xylitol and sorbitol demonstrated a significant dose-dependent inhibitory effect on S. mutans biofilms, whereas inhibitory effect on C. albicans biofilm was weak. The presence of 1 % sucrose in the environment diminished the inhibitory effect of both xylitol and sorbitol on S. mutans and C. albicans mono-species biofilms. Sucrose supplementation on pre-formed S. mutans biofilms also reduced the inhibitory effect of xylitol. Xylitol and sorbitol presence reduced mixed-species biofilm formation and altered the biofilm architecture and glucan production. However, sucrose supplementation reduced the inhibitory effect of sugar alcohols and enhanced the mixed-species biofilm formation. CONCLUSIONS: Xylitol and sorbitol exerts an inhibitory effect on S. mutans and C. albicans biofilm formation and this inhibitory effect is repressed by the presence of sucrose.


Assuntos
Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Sorbitol/farmacologia , Streptococcus mutans/efeitos dos fármacos , Sacarose/farmacologia , Xilitol/farmacologia
6.
Sci Rep ; 10(1): 15511, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968182

RESUMO

Osmolytes are organic solutes that change the protein folding landscape shifting the equilibrium towards the folded state. Herein, we use osmolytes to probe the structuring and aggregation of the intrinsically disordered mutant Huntingtin (mHtt) vis-a-vis the pathogenicity of mHtt on transcription factor function and cell survival. Using an inducible PC12 cell model of Huntington's disease (HD), we show that stabilizing polyol osmolytes drive the aggregation of Htt103QExon1-EGFP from a diffuse ensemble into inclusion bodies (IBs), whereas the destabilizing osmolyte urea does not. This effect of stabilizing osmolytes is innate, generic, countered by urea, and unaffected by HSP70 and HSC70 knockdown. A qualitatively similar result of osmolyte-induced mHtt IB formation is observed in a conditionally immortalized striatal neuron model of HD, and IB formation correlates with improved survival under stress. Increased expression of diffuse mHtt sequesters the CREB transcription factor to repress CREB-reporter gene activity. This repression is mitigated either by stabilizing osmolytes, which deplete diffuse mHtt or by urea, which negates protein-protein interaction. Our results show that stabilizing polyol osmolytes promote mHtt aggregation, alleviate CREB dysfunction, and promote survival under stress to support the hypothesis that lower molecular weight entities of disease protein are relevant pathogenic species in neurodegeneration.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Animais , Autofagia , Técnicas de Silenciamento de Genes , Glicerol/farmacologia , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteína Huntingtina/genética , Mutação , Concentração Osmolar , Células PC12 , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Dobramento de Proteína , Ratos , Sorbitol/farmacologia , Sacarose/farmacologia , Trealose/farmacologia
7.
Commun Biol ; 3(1): 528, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968183

RESUMO

Polyol and sugar osmolytes are commonly used in therapeutic protein formulations. How they may affect protein structure and function is an important question. In this work, through NMR measurements, we show that glycerol and sorbitol (polyols), as well as glucose (sugar), can shorten protein backbone hydrogen bonds. The hydrogen bond shortening is also captured by molecular dynamics simulations, which suggest a hydrogen bond competition mechanism. Specifically, osmolytes weaken hydrogen bonds between the protein and solvent to strengthen those within the protein. Although the hydrogen bond change is small, with the average experimental cross hydrogen bond 3hJNC' coupling of two proteins GB3 and TTHA increased by ~ 0.01 Hz by the three osmolytes (160 g/L), its effect on protein function should not be overlooked. This is exemplified by the PDZ3-peptide binding where several intermolecular hydrogen bonds are formed and osmolytes shift the equilibrium towards the bound state.


Assuntos
Glucose/farmacologia , Glicerol/farmacologia , Ligação de Hidrogênio/efeitos dos fármacos , Proteínas/efeitos dos fármacos , Sorbitol/farmacologia , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Pressão Osmótica , Ligação Proteica/efeitos dos fármacos , Proteínas/química
8.
Chem Pharm Bull (Tokyo) ; 68(7): 635-652, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32611999

RESUMO

Sodium-glucose cotransporter 1 (SGLT1) is the primary transporter for glucose absorption from the gastrointestinal tract. While C-phenyl D-glucitol derivative SGL5213 has been reported to be a potent intestinal SGLT1 inhibitor for use in the treatment of type 2 diabetes, no SGLT1 selectivity was found in vitro (IC50 29 nM for hSGLT1 and 20 nM for hSGLT2). In this study we found a new method of synthesizing key intermediates 12 by a one-pot three-component condensation reaction and discovered C-phenyl D-glucitol 41j (TP0454614), which has >40-fold SGLT1 selectivity in vitro (IC50 26 nM for hSGLT1 and 1101 nM for hSGLT2). The results of our study have provided new insights into the structure-activity relationships (SARs) of the SGLT1 selectivity of C-glucitol derivatives.


Assuntos
Transportador 1 de Glucose-Sódio/antagonistas & inibidores , Sorbitol/farmacologia , Animais , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Transportador 1 de Glucose-Sódio/metabolismo , Sorbitol/análogos & derivados , Sorbitol/química , Relação Estrutura-Atividade
9.
Obesity (Silver Spring) ; 28(5): 870-881, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32187881

RESUMO

OBJECTIVE: The aim of this study was to explore the dose response of licogliflozin, a dual inhibitor of sodium/glucose cotransporter 1 (SGLT1) and 2 (SGLT2), by evaluating change in body weight in adults with overweight or obesity. METHODS: This dose-response analysis evaluated change in body weight following 24 weeks with four once-daily and twice-daily licogliflozin doses (2.5-150 mg) versus placebo (primary end point). A further 24-week analysis evaluated the efficacy and safety of two once-daily licogliflozin doses in maintaining initial weight reduction. RESULTS: Licogliflozin once daily or twice daily produced a significant dose-response signal for weight loss versus placebo (P < 0.0001). However, mean adjusted percent changes in body weight after 24 weeks were modest, ranging from -0.45% to -3.83% (in the 50 mg twice daily group [95% CI: -5.26% to -2.48%]; n = 75). Responder analysis of ≥ 5% weight loss at week 24 revealed significant differences versus placebo, which were most pronounced with highest doses of 50 mg twice daily (45.3%) and 150 mg once daily (42.9%) (both P < 0.01). While weight loss was greater at higher doses, gastrointestinal adverse events were also more frequent. The 50-mg once-daily dose had perhaps the best balance between efficacy and tolerability. CONCLUSIONS: Licogliflozin produced significant reductions in body weight versus placebo. However, the magnitude of weight reduction was modest.


Assuntos
Anidridos/uso terapêutico , Peso Corporal/efeitos dos fármacos , Obesidade/tratamento farmacológico , Sobrepeso/tratamento farmacológico , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Sorbitol/análogos & derivados , Perda de Peso/efeitos dos fármacos , Adolescente , Adulto , Idoso , Anidridos/farmacologia , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Sorbitol/farmacologia , Sorbitol/uso terapêutico , Adulto Jovem
10.
Int J Mol Sci ; 21(5)2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32143531

RESUMO

The goal of this study was to examine the effect of lipid emulsion on the vasodilation induced by ATP-sensitive potassium (KATP) channels in isolated rat aortae and the underlying mechanism. The effects of Intralipid, containing 100% long-chain fatty acids, and Lipofundin MCT/LCT, containing 50% long-chain fatty acids plus 50% medium-chain fatty acids, on the vasodilation induced by levcromakalim in endothelium-intact aorta with or without NW-nitro-L-arginine methyl ester (L-NAME) and in endothelium-denuded aorta were examined. The effects of L-arginine, L-NAME, glibenclamide, and Lipofundin MCT/LCT, alone or combined, on the levcromakalim-induced vasodilation were examined. Lipofundin MCT/LCT inhibited the levcromakalim-induced vasodilation of isolated endothelium-intact aortae, whereas Intralipid did not. In addition, Lipofundin MCT/LCT had no effect on the levcromakalim-induced vasodilation of endothelium-denuded rat aortae and endothelium-intact aortae with L-NAME. L-arginine and Lipofundin MCT/LCT produced more levcromakalim-induced vasodilation than Lipofundin MCT/LCT alone. Glibenclamide inhibited levcromakalim-induced vasodilation. Levcromakalim did not significantly alter endothelial nitric oxide synthase phosphorylation, whereas Lipofundin MCT/LCT decreased cyclic guanosine monophosphate. Lipofundin MCT/LCT did not significantly alter levcromakalim-induced membrane hyperpolarization. Taken together, these results suggest that Lipofundin MCT/LCT inhibits the vasodilation induced by levcromakalim by inhibiting basally released endothelial nitric oxide, which seems to occur through medium-chain fatty acids.


Assuntos
Ácidos Graxos/química , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfolipídeos/farmacologia , Sorbitol/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Aorta/metabolismo , Cromakalim , GMP Cíclico/metabolismo , Combinação de Medicamentos , Emulsões , Células Endoteliais/metabolismo , Masculino , Potenciais da Membrana , Fosforilação , Canais de Potássio/metabolismo , Ratos , Ratos Sprague-Dawley
11.
Commun Biol ; 3(1): 145, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32218501

RESUMO

Yeast Snf1 (Sucrose non-fermenting1), mammalian AMPK (5' AMP-activated protein kinase) and plant SnRK1 (Snf1-Related Kinase1) are conserved heterotrimeric kinase complexes that re-establish energy homeostasis following stress. The hormone abscisic acid (ABA) plays a crucial role in plant stress response. Activation of SnRK1 or ABA signaling results in overlapping transcriptional changes, suggesting these stress pathways share common targets. To investigate how SnRK1 and ABA interact during stress response in Arabidopsis thaliana, we screened the SnRK1 complex by yeast two-hybrid against a library of proteins encoded by 258 ABA-regulated genes. Here, we identify 125 SnRK1- interacting proteins (SnIPs). Network analysis indicates that a subset of SnIPs form signaling modules in response to abiotic stress. Functional studies show the involvement of SnRK1 and select SnIPs in abiotic stress responses. This targeted study uncovers the largest set of SnRK1 interactors, which can be used to further characterize SnRK1 role in plant survival under stress.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Plantas Geneticamente Modificadas/efeitos dos fármacos , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Cloreto de Sódio/farmacologia , Sorbitol/farmacologia , Estresse Fisiológico , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , Pressão Osmótica , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Proteínas Serina-Treonina Quinases/genética , Estresse Salino , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido
12.
Appl Microbiol Biotechnol ; 104(8): 3529-3540, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32103313

RESUMO

Malassezia globosa is an opportunistic pathogen that causes various skin disorders, which disturbs people's life all the time, and conventional drugs are not completely satisfactory. Bacillomycin D (BD), an antifungal lipopeptide, could inhibit various fungi growth. However, the reports about its effect on M. globosa were not found yet. In this study, we showed that BD and BD-C16 (fatty acid chain had sixteen carbon atoms) completely inhibited growth of M. globosa at concentration of 64 µg/ml in 15 h, which was confirmed with the observation of irregular morphological change of M. globosa treated with BD. Significantly, the study on the working mechanism showed that BD induced cell death by changing cell membrane permeability and thus promoting the release of cellular contents, which may be mediated by the interaction between BD and ergosterol from membrane. Further study showed that BD reduced the overall content of cellular sterol, and interestingly, the expression of some genes involved in membrane and ergosterol synthesis were significantly upregulated, which was likely to be a feedback regulation. Besides, we found that BD had additive and synergistic effects with ketoconazole and amphotericin B, respectively, on inhibition of M. globosa, suggesting that combination use of BD with other commercial drugs could be a promising strategy to relieve skin disorders caused by M. globosa. KEY POINTS: • BD could efficiently inhibit the growth of M. globosa. • BD increases cell membrane permeability and thus promotes the release of cellular contents. • BD has additive or synergistic effect with other antifungal drugs.


Assuntos
Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Malassezia/efeitos dos fármacos , Malassezia/crescimento & desenvolvimento , Ergosterol/farmacologia , Testes de Sensibilidade Microbiana , Sorbitol/farmacologia
13.
Diabetologia ; 63(3): 577-587, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31897526

RESUMO

AIMS/HYPOTHESIS: Sodium-glucose cotransporter 2 (SGLT2) inhibitors, which prevent the renal reabsorption of glucose, decrease blood glucose levels in an insulin-independent manner. We previously reported creating a mouse model of systemic inhibition of target receptors for both insulin and IGF-1 by treating animals with OSI-906, a dual insulin/IGF-1 receptor inhibitor, for 7 days. The OSI-906-treated mice exhibited an increased beta cell mass, hepatic steatosis and adipose tissue atrophy, accompanied by hyperglycaemia and hyperinsulinaemia. In the present study, we investigated the effects of an SGLT2 inhibitor, luseogliflozin, on these changes in OSI-906-treated mice. METHODS: We treated C57BL/6J male mice either with vehicle, luseogliflozin, OSI-906 or OSI-906 plus luseogliflozin for 7 days, and phenotyping was performed to determine beta cell mass and proliferation. Subsequently, we tested whether serum-derived factors have an effect on beta cell proliferation in genetically engineered beta cells, mouse islets or human islets. RESULTS: SGLT2 inhibition with luseogliflozin significantly ameliorated hyperglycaemia, but not hyperinsulinaemia, in the OSI-906-treated mice. Liver steatosis and adipose tissue atrophy induced by OSI-906 were not altered by treatment with luseogliflozin. Beta cell mass and proliferation were further increased by SGLT2 inhibition with luseogliflozin in the OSI-906-treated mice. Luseogliflozin upregulated gene expression related to the forkhead box M1 (FoxM1)/polo-like kinase 1 (PLK1)/centromere protein A (CENP-A) pathway in the islets of OSI-906-treated mice. The increase in beta cell proliferation was recapitulated in a co-culture of Irs2 knockout and Insr/IR knockout (ßIRKO) beta cells with serum from both luseogliflozin- and OSI-906-treated mice, but not after SGLT2 inhibition in beta cells. Circulating factors in both luseogliflozin- and OSI-906-treated mice promoted beta cell proliferation in both mouse islets and cadaveric human islets. CONCLUSIONS/INTERPRETATION: These results suggest that luseogliflozin can increase beta cell proliferation through the activation of the FoxM1/PLK1/CENP-A pathway via humoral factors that act in an insulin/IGF-1 receptor-independent manner.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Sorbitol/análogos & derivados , Animais , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Sinergismo Farmacológico , Técnicas de Inativação de Genes , Humanos , Imidazóis/farmacologia , Proteínas Substratos do Receptor de Insulina/genética , Células Secretoras de Insulina/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Pirazinas/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/fisiologia , Receptor de Insulina/antagonistas & inibidores , Receptor de Insulina/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sorbitol/farmacologia
14.
Protein Pept Lett ; 27(3): 210-218, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31566125

RESUMO

BACKGROUND: Flagellin of Salmonella enterica serovar Enteritidis (SEF) stimulates immune responses to both itself and coapplied antigens. It is therefore used in vaccine development and immunotherapy. Removal of pathogenic S. enterica ser. Enteritidis from SEF production process is advantageous due to the process safety improvement. The protein solubility analysis using SDS-PAGE indicated that 53.49% of SEF expressed in Escherichia coli formed inclusion bodies. However, the protein recovery from inclusion bodies requires a complex process with a low yield. OBJECTIVE: We thus aim to study possibility of enhancing SEF expression in E. coli in soluble form using chemical and molecular chaperones. METHODS: Chemical chaperones including arginine, sorbitol, trehalose, sodium chloride and benzyl alcohol were used as cultivation medium additives during SEF expression. SEF solubilization by coexpression of molecular chaperones DnaK, DnaJ, and GrpE was also investigated. RESULTS: All of the chemical chaperones were effective in improving SEF solubility. However, sorbitol showed the most profound effect. SEF solubilization by molecular chaperones was slightly better than that using sorbitol and this approach enhanced noticeably SEF soluble concentration and SEF solubility percentage to almost two folds and 96.37% respectively. Results of limited proteolysis assay and native PAGE indicated similar conformational states and proper folding for SEF obtained without using chaperones and for those obtained using sorbitol and the molecular chaperones. However, the molecular chaperones based system was less costly than the sorbitol based system. CONCLUSION: The coexpression of molecular chaperones was then considered as the most appropriate approach for soluble SEF production. Therefore, SEF production for medical purposes is expected to be facilitated.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Flagelina/química , Chaperonas Moleculares/metabolismo , Salmonella enteritidis/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flagelina/genética , Flagelina/metabolismo , Corpos de Inclusão/química , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Salmonella enteritidis/química , Solubilidade , Sorbitol/farmacologia
15.
Mol Biol Cell ; 31(4): 287-303, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31851579

RESUMO

Eisosomes are membrane furrows at the cell surface of yeast that have been shown to function in two seemingly distinct pathways, membrane stress response and regulation of nutrient transporters. We found that many stress conditions affect both of these pathways by changing plasma membrane tension and thus the morphology and composition of eisosomes. For example, alkaline stress causes swelling of the cell and an endocytic response, which together increase membrane tension, thereby flattening the eisosomes. The flattened eisosomes affect membrane stress pathways and release nutrient transporters, which aids in their down-regulation. In contrast, glucose starvation or hyperosmotic shock causes cell shrinking, which results in membrane slack and the deepening of eisosomes. Deepened eisosomes are able to trap nutrient transporters and protect them from rapid endocytosis. Therefore, eisosomes seem to coordinate the regulation of both membrane tension and nutrient transporter stability.


Assuntos
Membrana Celular/metabolismo , Proteínas do Citoesqueleto/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Transporte de Nucleotídeos/genética , Fosfoproteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Proteínas do Citoesqueleto/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Proteínas de Transporte de Nucleotídeos/metabolismo , Pressão Osmótica , Fosfoproteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sorbitol/farmacologia , Tensão Superficial
16.
J Biol Chem ; 295(3): 673-689, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31780563

RESUMO

Insoluble, hyperubiquitylated TAR DNA-binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG-forming conditions induced very strong TDP-43 ubiquitylation, insolubility, and reduced splicing activity. Osmotic stress-induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R-like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal-regulated protein kinase modules and glycogen synthase kinase 3ß. For arsenite, but not for sorbitol, quenching oxidative stress with N-acetylcysteine did suppress both SG formation and TDP-43 ubiquitylation and insolubility. Thus, sodium arsenite appears to promote SG formation and TDP-43 modifications via oxidative stress, but sorbitol stimulates TDP-43 ubiquitylation and insolubility via a novel pathway(s) independent of SG formation. In conclusion, pathological TDP-43 modifications can be mediated via multiple distinct pathways for which SGs are not essential.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico/genética , Estresse Oxidativo/genética , Ubiquitinação/genética , Acetilcisteína/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Proteínas de Ligação a DNA/química , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Glicogênio Sintase Quinase 3 beta/genética , Células HEK293 , Proteínas de Choque Térmico/química , Humanos , Indóis/farmacologia , Mutação/efeitos dos fármacos , Pressão Osmótica/efeitos dos fármacos , Agregação Patológica de Proteínas/genética , Transporte Proteico/genética , Transdução de Sinais/efeitos dos fármacos , Solubilidade/efeitos dos fármacos , Sorbitol/farmacologia
17.
Biochem Biophys Res Commun ; 523(1): 54-59, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-31831169

RESUMO

Promotion of erythropoietin (EPO) production is important for erythropoiesis as well as cell viability. The most effective inducing factor for EPO production is hypoxia. Hypoxia inducible factor (HIF), a regulator of EPO production, is increased under hypoxic conditions and is also affected by various regulators such as sirtuin1 (SIRT1). SIRT1 is regulated by the cytoplasmic redox state, which is thought to affect EPO production. Therefore, we investigated the effects of sorbitol and lactic acid, which serve as substrates for cellular respiration and bring cells into a reduced state, on EPO production in HepG2 cells. The addition of low-concentration sorbitol to HepG2 cells produced a mildly reduced state similar to that of hypoxia and increased NAD+, SIRT1, and HIF-α, and EPO mRNA expression. On the other hand, lactate suppressed EPO mRNA expression at all concentrations. Inhibition of lactate production from pyruvate abolished the effect of low sorbitol concentrations on EPO mRNA expression. When low-concentration sorbitol and a reducing agent were administered simultaneously, the effect of increasing EPO mRNA expression disappeared. It was suggested that SIRT1 and EPO production increased under conditions where lactate production was not suppressed, even under mildly reduced conditions similar to hypoxia.


Assuntos
Eritropoetina/biossíntese , Ácido Láctico/farmacologia , Sorbitol/farmacologia , Animais , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Eritropoetina/genética , Células Hep G2 , Humanos , Ácido Láctico/administração & dosagem , Masculino , Oxirredução , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Sorbitol/administração & dosagem , Relação Estrutura-Atividade
18.
Lipids ; 55(1): 45-52, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31838756

RESUMO

Lipofundin is the solvent for propofol in the intravenous injection of Propofol-Lipuro® and is used in patients who need intravenous feeding to provide fatty acids and fat for energy. In addition to propofol, Lipofundin also affects the immune modulation of phagocytes. In a previous study, we reported that intravenous propofol effectively decreased Staphylococcus aureus-stimulated reactive oxygen species (ROS) levels, IL-1ß secretion, and phagocytosis in RAW264.7 macrophages. It is important to separately assess the effects of pure propofol, Lipofundin, and Propofol-Lipuro. By using an S. aureus-infected RAW264.7 macrophage model, the levels of secreted IL-1ß in cell supernatants were determined by ELISA. IL-1ß mRNA in cell pellets was further analyzed by quantitative polymerase chain reaction (qPCR), and Western blotting was performed to detect pro-IL-1ß synthesis. Total ROS levels were determined by a luminol chemiluminescence assay. Compared with pure propofol, treatment with clinically relevant concentrations of Propofol-Lipuro and Lipofundin obviously reduced IL-1ß secretion (>85% inhibition), S. aureus-stimulated ROS production (50% inhibition), and phagocytosis (>60% inhibition) to similar levels. Treatment with pure propofol alone significantly decreased IL-1ß mRNA levels and pro-IL-1ß protein synthesis, and slightly inhibited phagocytosis. In contrast, treatment with Propofol-Lipuro did not influence IL-1ß mRNA or pro-IL-1ß protein expression, even though treatment with Lipofundin increased the levels of both IL-1ß mRNA and its precursor protein. In conclusion, IL-1ß secretion is regulated at the posttranslational level. Lipofundin mediated the major effect of Propofol-Lipuro on the inhibition of IL-1ß secretion, ROS production, and phagocytosis in S. aureus-infected RAW264.7 cells.


Assuntos
Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/microbiologia , Fosfolipídeos/farmacologia , Propofol/farmacologia , Sorbitol/farmacologia , Staphylococcus aureus/imunologia , Administração Intravenosa , Animais , Regulação para Baixo , Combinação de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Fagocitose/efeitos dos fármacos , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo
19.
Arch Biochem Biophys ; 679: 108207, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31760123

RESUMO

PURPOSE: Obesity is a global health problem associated with several diseases including ocular complications. Earlier we reported progressive retinal degeneration because of obesity in a spontaneous obese rat (WNIN/Ob) model. In the current study, we examined the molecular mechanisms leading to retinal degeneration in WNIN/Ob rat. METHODS: Sorbitol was estimated by the fluorometric method in the retina of WNIN/Ob rats at different age (3-, 6- and 12- months), along with their respective lean rats. Immunoblotting was performed in the retina to assess the status of the insulin signaling pathway, ER stress and cellular stress (p38MAPK and ERK1/2). Human SK-N-SH cells were treated with 0.5 and 1.0 M sorbitol for 30 min to study insulin signaling, ER stress, and cellular stress. TUNEL assay was done to measure apoptosis. The retinal function in the rats was determined by electroretinogram. RESULTS: A gradual but significantly higher intracellular sorbitol accumulation was observed in the retina of obese rats from 3- to 12-months. The cellular osmotic stress has activated the insulin signaling mechanism without activating AKT and also triggered ER stress. Both the stresses activated the ERK and p38MAPK signaling causing apoptosis in the retina leading to retinal degeneration. Retinal dysfunction was confirmed by altered scotopic and photopic electroretinogram responses. These in vivo results were mimicked in SK-N-SH cells when exposed to sorbitol in vitro. CONCLUSIONS: These results suggest cellular stress due to sorbitol accumulation impairing the ER function, thereby leading to progressive retinal degeneration under obese conditions.


Assuntos
Estresse do Retículo Endoplasmático , Obesidade/patologia , Retina/patologia , Sorbitol/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Obesidade/metabolismo , Ratos , Receptor de Insulina/metabolismo , Retina/fisiopatologia , Sorbitol/farmacologia
20.
Fungal Genet Biol ; 135: 103289, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31704368

RESUMO

The HOG (high-osmolarity glycerol) pathway is critical for the appropriate adaptation to adverse conditions. Here, we demonstrated that the deletion of CgHog1 resulted in enhanced sensitivity to osmotic stress and increased resistance to fludioxonil in the poplar anthracnose fungus Colletotrichum gloeosporioides. The accumulation of chitin around hyphal tips was obviously decreased in the ΔCgHog1 strain under sorbitol, whereas it strongly was increased in the response to fludioxonil compared with the wild type. To investigate the underlying mechanism of CgHog1-mediated adaption to osmotic stress and fludioxonil, transcriptomic profiles were performed in both the ΔCgHog1 strain and the wild type under the treatment of sorbitol and fludioxonil, respectively. Under sorbitol, genes associated with glycolysis, lipid metabolism, and accumulation of soluble sugars and amino acids were differentially expressed; under fludioxonil, vesicle trafficking-related genes were highly downregulated in the ΔCgHog1 strain, which was consistent with abnormal vacuoles distribution and morphology of hyphae, indicating that the growth defect caused by fludioxonil may be associated with disruption of endocytosis. Taken together, we elucidated the adaptation mechanisms of how CgHog1 regulates appropriate response to sorbitol and fludioxonil via different metabolism pathways. These findings extend our insights into the HOG pathway in fungi.


Assuntos
Colletotrichum/efeitos dos fármacos , Colletotrichum/genética , Dioxóis/farmacologia , Proteínas Fúngicas/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Pirróis/farmacologia , Sorbitol/farmacologia , Adaptação Fisiológica , Regulação para Baixo , Regulação Fúngica da Expressão Gênica , Hifas/metabolismo , Pressão Osmótica , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Populus/microbiologia
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