RESUMO
Cobalt chloride can create hypoxia-like state in vitro (referred to as chemical hypoxia). Several studies have suggested that chemical hypoxia may cause deleterious effects on myogenesis. The intrinsic underlying mechanisms of myoblast differentiation, however, are not fully understood. Here, we show that cobalt chloride strongly suppresses myoblast differentiation in a dose-dependent manner. The impaired myoblast differentiation is accompanied by downregulation of myogenic regulatory factor myogenin. Under chemical hypoxia, myogenin stability is decreased at mRNA and protein levels. A muscle-specific E3 ubiquitin ligase MAFbx, which can target myogenin protein for proteasomal degradation, is upregulated along with changes in Akt/Foxo and AMPK/Foxo signaling pathways. A proteasome inhibitor completely prevents cobalt chloride-mediated decrease in myogenin protein. These results suggest that cobalt chloride might modulate myogenin expression at post-transcriptional and post-translational levels, resulting in the failure of the myoblasts to differentiate into myotubes.
Assuntos
Hipóxia Celular , Cobalto/farmacologia , Regulação para Baixo , Mioblastos/citologia , Miogenina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Camundongos , Desenvolvimento Muscular , Mioblastos/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
The active form of vitamin D (1α, 25-dihydroxyvitamin D3 [1α, 25(OH)2D3], referred to as 1,25D) has been suggested to play a pivotal role in skeletal muscle function and metabolism. However, the mechanisms through which 1,25D functions in this tissue remain to be elucidated. Recent studies have shown that vitamin D signaling regulates neuromuscular maintenance and improves locomotion in mice. In the present study, we examined the effects of 1,25D on neuromuscular synaptogenesis by measuring clustering of acetylcholine receptors (AChRs) in C2C12 myotubes. 1,25D treatment enhanced the agrin-induced AChR clustering in myotubes compared to treatment with agrin alone. Furthermore, siRNA-mediated knockdown of the vitamin D receptor (VDR) decreased the agrin-induced AChR clustering. 1,25D increased the expression of rapsyn, which is necessary for AChR clustering, while demonstrating no effect on other neuromuscular junction-related genes. In addition, rapsyn expression was dependent on 1,25D-VDR signaling. These results suggest that 1,25D-VDR signaling may regulate rapsin expression, resulting in the up-regulation of agrin-induced AChR clustering.
RESUMO
Spinal muscular atrophy (SMA) is a severe genetic neuromuscular disorder caused by insufficiency of functional survival motor neuron (SMN) protein. Several clinical trials have been conducted with the aim of upregulating the expression of the SMN protein in SMA patients. In order to evaluate the efficiency of these SMN-targeted approaches, it has become necessary to verify SMN protein levels in the cells of SMA patients. Accordingly, we have developed a method allowing the evaluation of the functional SMN protein with < 1.5 mL of peripheral blood using imaging flow cytometry. The expression of SMN protein in CD3+, CD19+, and CD33++ cells obtained from SMA patients, was significantly reduced compared with that in cells from control subjects. In spot analysis of CD33++ cells, the intensities of SMN spots were significantly reduced in SMA subjects, when compared with that in controls. Therefore, SMN spots implied the presence of functional SMN protein in the cell nucleus. To our knowledge, our results are the first to demonstrate the presence of functional SMN protein in freshly isolated peripheral blood cells. We anticipate that SMN spot analysis will become the primary endpoint assay for the evaluation and monitoring of therapeutic intervention, with SMN serving as a reliable biomarker of therapeutic efficacy in SMA patients.
Assuntos
Atrofia Muscular Espinal/sangue , Proteína 1 de Sobrevivência do Neurônio Motor/sangue , Adolescente , Adulto , Antígenos CD19/sangue , Biomarcadores/sangue , Complexo CD3/sangue , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/sangue , Proteína 2 de Sobrevivência do Neurônio Motor/sangue , Adulto JovemRESUMO
BACKGROUND: Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose. METHODS: After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein. RESULTS: Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. CONCLUSIONS: The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials.
Assuntos
Citometria de Fluxo , Perfilação da Expressão Gênica , Atrofia Muscular Espinal/metabolismo , Transcriptoma , Pré-Escolar , Variações do Número de Cópias de DNA , Feminino , Humanos , Imuno-Histoquímica , Linfócitos/metabolismo , Linfócitos/patologia , Masculino , Atrofia Muscular Espinal/patologia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Spinal muscular atrophy (SMA) is caused by mutations within the survival motor neuron 1 (SMN1) gene. These mutations result in the reduction of survival motor neuron (SMN) protein expression and SMN complex in spinal motor neurons and other tissues. SMN protein has been used as a therapeutic biomarker in recent SMA clinical studies using enzyme-linked immunosorbent assay (ELISA). Here, we investigated whether imaging flow cytometry can be a viable source of quantitative information on the SMN protein. Using a FlowSight imaging flow cytometer (Merck-Millipore, Germany), we demonstrated that imaging flow cytometry could successfully identify different expression patterns and subcellular localization of SMN protein in healthy human fibroblasts and SMA patient-derived fibroblasts. In addition, we could also evaluate the therapeutic effects of SMN protein expression by valproic acid treatment of SMA patient-derived cells in vitro. Therefore, we suggest that imaging flow cytometry technology has the potential for identifying SMN protein expression level and pattern as an evaluation tool of clinical studies.
Assuntos
Biomarcadores/metabolismo , Citometria de Fluxo/métodos , Atrofia Muscular Espinal/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Sequência de Bases , Células Cultivadas , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/metabolismoRESUMO
OBJECTIVE: To assess the utility of liraglutide, a GLP-1 receptor agonist, as additional therapy following resolution of glucotoxicity with insulin therapy. METHODS: The subjects were 13 Japanese patients with short-duration type 2 diabetes mellitus (2.0 ± 2.1 years). At first, treatment with insulin therapy consisted of bolus insulin before each meal and basal insulin at bed time commenced to improve every preprandial glucose levels below 130 mg/dL. Then, insulin therapy was replaced with liraglutide monotherapy in case in which 50% or more self-monitoring of blood glucose (SMBG) tests revealed preprandial glucose levels of less than 130 mg/dL at least for one month. Liraglutide dosing was initiated at 0.3 mg/day and increased in weekly or biweekly increments of 0.3 mg/day, to the maximum permissible dose (in Japan) of 0.9 mg/day. The participants were treated with liraglutide for 24 weeks. RESULTS: The average insulin therapy period was 13.2 ± 5.4 weeks, and insulin therapy significantly improved HbA1c values from 12.4% ± 1.6% to 6.8% ± 0.9% (P < 0.05). After improvement of hyperglycemia with insulin therapy and switching to liraglutide monotherapy for 24 weeks, HbA1c values remained constant (6.2% ± 1.0% at week 24) and the rates of hypoglycemic episodes significantly decreased (P < 0.05). CONCLUSIONS: These data suggest that liraglutide is proposed as an alternative follow-up therapy subsequent to eliminate glucotoxicity with insulin therapy.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Hemoglobinas Glicadas/efeitos dos fármacos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Receptores de Glucagon/agonistas , Adulto , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Automonitorização da Glicemia , Diabetes Mellitus Tipo 2/sangue , Feminino , Seguimentos , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Receptor do Peptídeo Semelhante ao Glucagon 1 , Hemoglobinas Glicadas/metabolismo , Humanos , Japão , Liraglutida , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
This is the first study to demonstrate sonoelectrocatalytic disinfection using titanium dioxide (TiO(2)) as an anode for effective inactivation of Escherichia coli. In brief, a non-woven TiO(2) fabric used as an anode and a platinum cathode were immersed in an E. coli suspension in which a positive potential was applied to TiO(2) concomitant with ultrasound (US) irradiation. Two control experiments were performed using E. coli suspensions to exhibit the effects of the sonoelectrocatalytic disinfection. One was disinfection by applying a positive potential to a TiO(2) electrode, but without US irradiation (electrochemical disinfection). The other was disinfection without applying a potential, but with US irradiation in the presence of TiO(2) (sonocatalytic disinfection). The cell inactivation rate in sonoelectrocatalytic disinfection was synergistically much more enhanced than the combined inactivation rates in electrochemical disinfection and sonocatalytic disinfection. This synergistically enhanced inactivation rate of E. coli cells was attributable to effective reaction of the sonocatalytically generated OH radicals with E. coli cells at the surface of the TiO(2) anode, which resulted from the electroadsorption of E. coli cells toward the TiO(2) anode.
Assuntos
Desinfecção/métodos , Eletrodos , Escherichia coli/efeitos dos fármacos , Titânio/farmacologia , Ultrassom , Adsorção , Catálise , Técnicas Eletroquímicas , Titânio/químicaRESUMO
Thrombosis is characterized by congenital and acquired procatarxis. Lactic acid bacteria-fermented soybean milk products (FS-LAB) inhibit hepatic lipid accumulation and prevent atherosclerotic plaque formation. However, the therapeutic efficacy of FS-LAB against thrombosis has yet to be investigated. In this study, FS-LAB were administered subcutaneously into the tails of rats, with the subsequent intravenous administration of κ-carrageenan 12 hr after the initial injection. In general, administration of κ-carrageenan induces thrombosis. The length of the infarcted tail regions was significantly shorter in the rats administered a single-fold or double-fold concentration of the FS-LAB solution compared with the region in control rats. Therefore, FS-LAB exhibited significant antithrombotic effects. Our study is the first to characterize the properties of FS-LAB and, by testing their efficacy on an in vivo rat model of thrombosis, demonstrate the potency of their antithrombotic effect.
RESUMO
The abuse of antibacterial drugs imposes a selection pressure on bacteria that has driven the evolution of multidrug resistance in many pathogens. Our efforts to discover novel classes of antibiotics to combat these pathogens resulted in the discovery of amycolamicin (AMM). The absolute structure of AMM was determined by NMR spectroscopy, X-ray analysis, chemical degradation, and modification of its functional groups. AMM consists of trans-decalin, tetramic acid, two unusual sugars (amycolose and amykitanose), and dichloropyrrole carboxylic acid. The pyranose ring named as amykitanose undergoes anomerization in methanol. AMM is a potent and broad-spectrum antibiotic against Gram-positive pathogenic bacteria by inhibiting DNA gyrase and bacterial topoisomerase IV. The target of AMM has been proved to be the DNA gyrase B subunit and its binding mode to DNA gyrase is different from those of novobiocin and coumermycin, the known DNA gyrase inhibitors.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/química , Glucosídeos/química , Glucosídeos/farmacologia , Pirróis/química , Pirróis/farmacologia , Inibidores da Topoisomerase II , Bactérias/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade MicrobianaRESUMO
We examined the influence of fruit juice on fingertips on self-monitoring of blood glucose (SMBG) results and patient behavior regarding SMBG. We found hand washing with tap water after touching the flesh of fruit, rather than using alcohol swabs, to be very important for accurate SMBG.
Assuntos
Bebidas , Automonitorização da Glicemia/métodos , Frutas , Humanos , Reprodutibilidade dos TestesRESUMO
First-trimester chorionic-villi-derived cells (FTCVs) are the earliest fetal material that can be obtained for prenatal diagnosis of fetal disorders such as Duchenne muscular dystrophy (DMD). DMD is a devastating X-linked disorder characterized by the absence of dystrophin at the sarcolemma of muscle fibers. Currently, a limited number of treatment options are available for DMD, although cell therapy is a promising treatment strategy for muscle degeneration in DMD patients. A novel candidate source of cells for this approach is FTCVs taken between the 9th and 11th weeks of gestation. FTCVs might have a higher undifferentiated potential than any other tissue-derived cells because they are the earliest fetal material. We examined the expression of mesenchymal stem cell and pluripotent stem cell markers in FTCVs, in addition to their myogenic potential. FTCVs expressed mesenchymal stem cell markers and Nanog and Sox2 transcription factors as pluripotent stem cell markers. These cells efficiently differentiated into myotubes after myogenic induction, at which point Nanog and Sox2 were down-regulated, whereas MyoD, myogenin, desmin and dystrophin were up-regulated. To our knowledge, this is the first demonstration that FTCVs can be efficiently directed to differentiate in vitro into skeletal muscle cells that express dystrophin as the last stage marker of myogenic differentiation. The myogenic potential of FTCVs reveals their promise for use in cell therapy for DMD, for which no effective treatment presently exists.
Assuntos
Vilosidades Coriônicas/metabolismo , Desenvolvimento Muscular , Primeiro Trimestre da Gravidez/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Distrofina/genética , Distrofina/metabolismo , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Microscopia de Contraste de Fase , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Proteína Homeobox Nanog , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Gravidez , Terceiro Trimestre da Gravidez/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The aim of this study was to investigate the role of insulin receptor substrate-2 (IRS-2) mediated signal in macrophages on the accumulation of macrophages in the vascular wall. Mice transplanted with IRS-2(-/-) bone marrow, a model of myeloid cell restricted defect of IRS-2, showed accumulation of monocyte chemoattractant protein-1-expressing macrophages in the vascular wall. Experiments using cultured peritoneal macrophages showed that IRS-2-mediated signal pathway stimulated by physiological concentrations of insulin, not by IL-4, contributed to the suppression of monocyte chemoattractant protein-1 expression induced by lipopolysaccharide. Our data indicated that IRS-2 deficiency in macrophages enhanced their accumulation in the vascular wall accompanied by increased expression of proinflammatory mediators in macrophages. These results suggest a role for insulin resistance in macrophages in early atherosclerogenesis.
Assuntos
Vasos Sanguíneos/imunologia , Proteínas Substratos do Receptor de Insulina/deficiência , Macrófagos/imunologia , Animais , Transplante de Medula Óssea , Quimiocina CCL2/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Glucagon-like peptide-1 is a hormone secreted by L cells of the small intestine and stimulates glucose-dependent insulin response. Glucagon-like peptide-1 receptor agonists such as exendin-4 are currently used in type 2 diabetes, and considered to have beneficial effects on the cardiovascular system. To further elucidate the effect of glucagon-like peptide-1 receptor agonists on cardiovascular diseases, we investigated the effects of exendin-4 on intimal thickening after endothelial injury. Under continuous infusion of exendin-4 at 24 nmol/kg/day, C57BL/6 mice were subjected to endothelial denudation injury of the femoral artery. Treatment of mice with exendin-4 reduced neointimal formation at 4weeks after arterial injury without altering body weight or various metabolic parameters. In addition, in vitro studies of isolated murine, rat and human aortic vascular smooth muscle cells showed the expression of GLP-1 receptor. The addition of 10nM exendin-4 to cultured smooth muscle cells significantly reduced their proliferation induced by platelet-derived growth factor. Our results suggested that exendin-4 reduced intimal thickening after vascular injury at least in part by the suppression of platelet-derived growth factor-induced smooth muscle cells proliferation.
Assuntos
Neointima/prevenção & controle , Peptídeos/uso terapêutico , Receptores de Glucagon/agonistas , Túnica Íntima/efeitos dos fármacos , Lesões do Sistema Vascular/tratamento farmacológico , Peçonhas/uso terapêutico , Animais , Peso Corporal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Exenatida , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1 , Glucose/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Ratos , Transdução de Sinais , Túnica Íntima/patologia , Lesões do Sistema Vascular/patologiaRESUMO
Thrombosis is characterized by congenital and acquired procatarxis. Nattokinase inhibits thrombus formation in vitro. However, in vivo evaluation of the therapeutic efficacy of nattokinase against thrombosis remains to be conducted. Subcutaneous nattokinase injections of 1 or 2 mg/ml were administered to the tails of rats. Subsequently, κ-carrageenan was intravenously administered to the tails at 12 h after nattokinase injections. The mean length of the infarcted regions in the tails of rats was significantly shorter in rats administered 2 mg/ml of nattokinase than those in control rats. Nattokinase exhibited significant prophylactic antithrombotic effects. Previously, the in vitro efficacy of nattokinase against thrombosis had been reported; now our study has revealed the in vivo efficacy of nattokinase against thrombosis.
Assuntos
Subtilisinas/uso terapêutico , Trombose/tratamento farmacológico , Animais , Carragenina , Modelos Animais de Doenças , Feminino , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Infarto/tratamento farmacológico , Ratos , Ratos Wistar , Alimentos de Soja , Cauda/irrigação sanguínea , Trombose/induzido quimicamente , Trombose/prevenção & controleRESUMO
We have successfully achieved transcutaneous immunization without the use of any skin pre-treatment or immune-stimulant adjuvant by applying a solid-in-oil (S/O) nanodispersion: an oil-based nanodispersion of antigens coated with hydrophobic surfactant molecules. This finding indicates that the S/O nanodispersion has great promise for effective transcutaneous vaccination.
Assuntos
Administração Cutânea , Coloides/química , Imunização/métodos , Óleos , Animais , Antígenos/administração & dosagem , Coloides/administração & dosagem , Cobaias , Interações Hidrofóbicas e Hidrofílicas , Camundongos , TensoativosRESUMO
OBJECTIVE: Exogenous administration of glucagon-like peptide-1 (GLP-1) or GLP-1 receptor agonists such as an exendin-4 has direct beneficial effects on the cardiovascular system. However, their effects on atherosclerogenesis have not been elucidated. The aim of this study was to investigate the effects of GLP-1 on accumulation of monocytes/macrophages on the vascular wall, one of the earliest steps in atherosclerogenesis. RESEARCH DESIGN AND METHODS: After continuous infusion of low (300 pmol . kg(-1) . day(-1)) or high (24 nmol . kg(-1) . day(-1)) dose of exendin-4 in C57BL/6 or apolipoprotein E-deficient mice (apoE(-/-)), we evaluated monocyte adhesion to the endothelia of thoracic aorta and arteriosclerotic lesions around the aortic valve. The effects of exendin-4 were investigated in mouse macrophages and human monocytes. RESULTS: Treatment with exendin-4 significantly inhibited monocytic adhesion in the aortas of C57BL/6 mice without affecting metabolic parameters. In apoE(-/-) mice, the same treatment reduced monocyte adhesion to the endothelium and suppressed atherosclerogenesis. In vitro treatment of mouse macrophages with exendin-4 suppressed lipopolysaccharide-induced mRNA expression of tumor necrosis factor-alpha and monocyte chemoattractant protein-1, and suppressed nuclear translocation of p65, a component of nuclear factor-kappaB. This effect was reversed by either MDL-12330A, a cAMP inhibitor or PKI(14-22), a protein kinase A-specific inhibitor. In human monocytes, exendin-4 reduced the expression of CD11b. CONCLUSIONS: Our data suggested that GLP-1 receptor agonists reduced monocyte/macrophage accumulation in the arterial wall by inhibiting the inflammatory response in macrophages, and that this effect may contribute to the attenuation of atherosclerotic lesion by exendin-4.
Assuntos
Aterosclerose/patologia , Adesão Celular/fisiologia , Endotélio Vascular/fisiologia , Monócitos/fisiologia , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Peçonhas/farmacologia , Peçonhas/uso terapêutico , Animais , Aorta/fisiologia , Aterosclerose/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Exenatida , Citometria de Fluxo , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Teste de Tolerância a Glucose , Humanos , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Fígado/efeitos dos fármacos , Fígado/fisiologia , Pulmão/efeitos dos fármacos , Pulmão/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Size reduction of drug particles to the nanoscale is important in improving the dissolution rate of poorly water-soluble drugs. The aim of this study was to investigate the physicochemical properties of griseofulvin (GF)-lipid nanoparticles and the interactions between GF-lipid nanoparticles and various saccharides during freeze-drying. The phase transition temperature of the GF-lipid nanoparticle suspension was 56.8 degrees C, whereas that of the lipid nanoparticle suspension alone was 57.9 degrees C, indicating that the GF crystals were incorporated into the lipid phase. The mean particle size of a rehydrated suspension of xylose-containing freeze-dried GF-lipid nanoparticles was about 220 nm. However, the mean particle size on the rehydration of nanoparticles containing mannose (monosaccharide), fructose (disaccharide), lactose (disaccharide), or raffinose (trisaccharide) was about 60 nm, suggesting that these saccharides prevented aggregation during the freeze-drying process. Powder X-ray diffraction revealed that xylose existed in the crystalline state in the freeze-dried nanoparticles, whereas the other saccharides existed in amorphous states. Thus, the crystallization of the saccharide was found to be strongly correlated with the aggregation property of the nanoparticles. In the case of freeze-dried xylose, the nanoparticles were squeezed out as the saccharine crystal lattice arranged itself regularly. Then, the ejected nanoparticles were aggregated. In contrast, in the case of the other freeze-dried saccharide, the saccharide remained incorporated with the GF-lipid nanoparticles because its crystal lattice was arranged irregularly. Thus, the particle size was maintained.
Assuntos
Fenômenos Químicos , Portadores de Fármacos/química , Griseofulvina/administração & dosagem , Lipídeos/química , Monossacarídeos/química , Nanopartículas/química , Oligossacarídeos/química , Cristalização , Composição de Medicamentos , Estabilidade de Medicamentos , Liofilização , Frutose/química , Lactose/química , Manose/química , Tamanho da Partícula , Difração de Pó , Rafinose/química , Suspensões , Difração de Raios XRESUMO
Beta-cell proliferation is regulated by various metabolic demands including peripheral insulin resistance, obesity, and hyperglycemia. In addition to enhancement of glucose-induced insulin secretion, agonists for glucagon-like peptide-1 receptor (GLP-1R) stimulate proliferation and inhibit apoptosis of beta-cells, thereby probably preserve beta-cell mass. To evaluate the beta-cell preserving actions of GLP-1R agonists, we assessed the acute and chronic effects of exendin-4 on beta-cell proliferation, mass and glucose tolerance in C57BL/6J mice under various conditions. Short-term administration of high-dose exendin-4 transiently stimulated beta-cell proliferation. Comparative transcriptomic analysis showed upregulation of IGF-1 receptor and its downstream effectors in islets. Treatment of mice with exendin-4 daily for 4 weeks (long-term administration) and feeding high-fat diet resulted in significant inhibition of weight gain and improvement of glucose tolerance with reduced insulin secretion and beta-cell mass. These findings suggest that long-term GLP-1 treatment results in insulin sensitization of peripheral organs, rather than enhancement of beta-cell proliferation and function, particularly when animals are fed high-fat diet. Thus, the effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation largely depend on treatment dose, duration of treatment and meal contents. While GLP-1 enhances proliferation of beta-cells in some diabetic mice models, our results suggest that GLP-1 stimulates beta-cell growth only when expansion of beta-cell mass is required to meet metabolic demands.
Assuntos
Hipoglicemiantes/administração & dosagem , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Peptídeos/administração & dosagem , Peçonhas/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Exenatida , Alimentos , Expressão Gênica/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1 , Glucose/metabolismo , Teste de Tolerância a Glucose , Hipoglicemiantes/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/farmacologia , Receptores de Glucagon/agonistas , Peçonhas/farmacologiaRESUMO
AIM: We have attempted to micronize drug particles with a particle size of less than 100 nm and maintain the particle size of their suspension to improve the solubility and bioavailability of poorly water-soluble drugs. Furthermore, the method of freeze-drying nanoparticles was applied to maintain particulate nature of nanoparticles containing various saccharides and sugar alcohols for a long time. METHOD: Griseofulvin (GF)-lipid nanoparticle suspension is prepared using GF and a lipid by high-pressure homogenization. The particle size of the obtained GF-lipid nanoparticle suspension is maintained constant by freeze-drying. RESULT: The mean particle size of GF-lipid nanoparticles prepared by high-pressure homogenization is approximately 60 nm. The mean particle size remains less than 100 nm for 1 month. The GF-lipid nanoparticle suspension containing xylitol, trehalose, or sucrose is freeze-dried to maintain the particulate nature. The mean particle size of the rehydrated suspension is lower than that of the rehydrated suspension containing erythritol or lactose. In particular, it is new knowledge to have found that an aggregation is minimized by adding xylitol which is sugar alcohol. The minimum concentration of xylitol, trehalose, and sucrose required to maintain a constant particle size by rehydration is 3%, 3%, and 5% (w/v), respectively.