RESUMO
BACKGROUND: Preß1-high-density lipoprotein (HDL) is a lipid-poor cholesterol acceptor that is converted to lipid-rich HDL by lecithin-cholesterol acyltransferase (LCAT). In patients receiving hemodialysis, preß1-HDL metabolism is hampered even if HDL cholesterol is normal. Hemodialysis may affect preß1-HDL metabolism by releasing lipases from the vascular wall due to heparin. OBJECTIVES: We investigated whether preß1-HDL metabolism is delayed in patients with chronic kidney disease (CKD) who are not receiving hemodialysis. METHODS: We examined 44 patients with Stage 3 or higher CKD and 22 healthy volunteers (Control group). The patients with CKD were divided into those without renal replacement therapy (CKD group, n = 22) and those undergoing continuous ambulatory peritoneal dialysis (CAPD group, n = 22). Plasma preß1-HDL concentrations were determined by immunoassay. During incubation at 37°C, we used 5,5-dithio-bis (2-nitrobenzoic acid) (DTNB) to inhibit LCAT activity and defined the conversion halftime of preß1-HDL (CHTpreß1) as the time required for the difference in preß1-HDL concentration in the presence and absence of 5,5-DTNB to reach half the baseline concentration. RESULTS: The absolute and relative preß1-HDL concentrations were higher, and CHTpreß1 was longer in the CKD and CAPD groups than in the Control group. Preß1-HDL concentration was significantly correlated with CHTpreß1 but not with LCAT activity in patients with CKD and CAPD. CONCLUSION: Preß1-HDL metabolism is delayed in patients with CKD who are not on hemodialysis. This preß1-HDL metabolic delay may progress as renal function declines.
Assuntos
Lipoproteínas de Alta Densidade Pré-beta/metabolismo , Diálise Renal/métodos , Insuficiência Renal Crônica/metabolismo , Terapia de Substituição Renal/métodos , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/terapiaRESUMO
BACKGROUND: Triglyceride (TG) is a tri-ester composed of a glycerol and 3 fatty acids. Degradation of TG in adipose tissue is increased in the fasting state but inhibited in the postprandial state. Although insulin suppresses adipose TG degradation, patients with insulin resistance have high concentrations of insulin and free glycerol (FG) in the fasting state. OBJECTIVE: We examined whether the fasting FG concentration reflects visceral obesity and insulin sensitivity in middle-aged Japanese men. METHODS: We measured the fasting serum FG concentration in 72 males aged 30 to 50 years using a simple enzymatic method. The subjects were divided into tertiles according to their homeostasis model assessment of insulin resistance (HOMA-IR). Besides routine glucose- and lipid-related parameters, we determined insulin sensitivity as the rate of glucose disappearance in a 2-step hyperinsulinemic-euglycemic clamp and the abdominal visceral fat area (VFA) by magnetic resonance imaging. RESULTS: The highest HOMA-IR tertile group had a higher fasting FG concentration than the middle- and lowest-tertile groups (0.077 ± 0.024 vs 0.063 ± 0.017 and 0.061 ± 0.016 mmol/L, P < .05 and P < .01). The FG concentration was positively correlated with VFA (rs = 0.36; P < .01) and the HOMA-IR score (rs = 0.26, P < .05) but negatively correlated with insulin sensitivity (rs = -0.26, P < .05). Multivariate regression analysis revealed that the FG concentration is independently associated with VFA and insulin sensitivity. CONCLUSION: The fasting FG concentration reflects VFA and insulin sensitivity in middle-aged Japanese men. The fasting FG concentration may be a potential surrogate marker of visceral obesity and insulin resistance in outpatients.
Assuntos
Jejum/sangue , Glicerol/sangue , Resistência à Insulina , Obesidade Abdominal/sangue , Tecido Adiposo/patologia , Adulto , Idoso , Biomarcadores/sangue , Glicemia/metabolismo , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Obesidade Abdominal/metabolismo , Obesidade Abdominal/patologia , Período Pós-Prandial , Triglicerídeos/sangueRESUMO
Serum iron concentration increases in marathon athletes after running due to mechanical destruction of red blood cells (hemolysis). This study was performed to examine whether serum iron concentration increases after regular Judo exercise, and if so, whether such post-exercise iron increase is caused by hemolysis. We examined biochemical parameters related to red blood cell and iron metabolism in 16 male competitive Judo athletes before and after traditional exercise training composed of basic movements and freestyle matchup. The parameters were adjusted for changes in plasma volume based on simultaneously measured albumin concentration. The red blood cell count, hemoglobin concentration, and hematocrit levels decreased significantly, by 6.0-8.4%, after Judo exercise. The serum iron concentration and transferrin saturation increased significantly, from 87 ± 34 µg/dL to 98 ± 29 µg/dL and from 27.1 ± 9.7% to 31.2 ± 9.0%, respectively. Furthermore, the serum free hemoglobin level increased by 33.9% (p < 0.05), and haptoglobin concentration decreased by 19.2% (p < 0.001). A significant negative correlation was observed between Δ haptoglobin concentration and Δ serum iron concentration (r = - 0.551, p = 0.027). The results of this study indicate that serum iron concentration increases significantly after Judo exercise due to hemolysis.
Assuntos
Ferro , Artes Marciais , Atletas , Exercício Físico , Hemoglobinas , Hemólise , Humanos , MasculinoRESUMO
BACKGROUND: High-density lipoprotein-cholesterol (HDL-C) is generally measured using several homogeneous assays. We aimed to clarify whether apolipoprotein E-containing HDL (apoE-HDL) subfractions are altered during storage, and if so, whether such changes affect the HDL-C concentration measured using homogeneous assays. METHODS: We stored serum from normolipidemic (n=32) and dyslipidemic (n=17) subjects at 4°C for up to 7days. ApoE-HDL subfractions were analyzed using native 2-dimensional gel (native 2D-gel) electrophoresis. HDL-C concentrations were determined using 2 precipitation and 4homogeneous assays. RESULTS: Native 2D-gel electrophoresis revealed variously sized apoE-HDL subfractions. After 4h incubation at 37°C, subfractions of smaller particles were converted into larger particles by lecithin:cholesterol acyltransferase (LCAT) activity. After 7days storage at 4°C, the smaller subfractions were decreased in the normolipidemic group, accompanying increases in larger subfractions, whereas changes in the respective subfractions varied among individuals in the dyslipidemic group. HDL-C concentrations were significantly lower after storage at 4°C in all assays, except that using Sekisui Medical's reagent. Therefore, changes in HDL-C concentration and apoE-HDL subfractions were independent of each other. CONCLUSION: ApoE-HDL subfractions change during storage, but these changes are not linked to those in HDL-C concentration measured using homogeneous assays.
Assuntos
Apolipoproteínas E/sangue , Análise Química do Sangue/métodos , HDL-Colesterol/sangue , Dislipidemias/sangue , Lipoproteínas HDL/sangue , Adulto , Coleta de Amostras Sanguíneas , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Temperatura , Adulto JovemRESUMO
BACKGROUND: Preß1-high-density lipoprotein (HDL) is an efficient acceptor of cell-derived free cholesterol, which is converted into lipid-rich HDL by lecithin-cholesterol acyltransferase. Previous studies have shown that preß1-HDL is significantly higher in individuals with hyperlipidemia. Preß1-HDL concentrations may be altered in smokers, who are at high risk for atherosclerosis. OBJECTIVE: The aim of the present study was to investigate the effect of smoking on preß1-HDL concentrations. METHODS: We measured the preß1-HDL concentration and lecithin-cholesterol acyltransferase-dependent conversion rate (CHTpreß1) in 74 men (39 nonsmokers and 35 smokers) using an immunoassay. RESULTS: The smoker and nonsmoker groups were further divided into normolipidemic and hyperlipidemic subjects. Among nonsmokers, the mean preß1-HDL concentration was 27% higher in hyperlipidemics than in normolipidemics (25.5 ± 6.7 vs 20.3 ± 4.6 mg/L apoAI, P < .01). In contrast, mean preß1-HDL concentrations did not differ between hyperlipidemic and normolipidemic smokers (19.9 ± 3.1 vs 22.4 ± 6.9 mg/L apoAI). We found a positive correlation between preß1-HDL concentration and CHTpreß1 in nonsmokers, but not in smokers. Smoking a single cigarette did not change preß1-HDL concentrations or CHTpreß1. Compared with nonsmokers, preß1-HDL concentrations were relatively low in hyperlipidemic smokers but not in normolipidemic smokers, and CHTpreß1 was not a significant determinant of preß1-HDL concentrations in smokers. CONCLUSION: Our findings suggest that smoking may be disadvantageous to individuals with hyperlipidemia because preß1-HDL metabolism is altered.
Assuntos
Hiperlipidemias/sangue , Lipoproteínas HDL/sangue , Fumar/efeitos adversos , Feminino , Humanos , Hiperlipidemias/enzimologia , Hiperlipidemias/etiologia , Lipoproteínas HDL/química , Masculino , Pessoa de Meia-Idade , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , RiscoRESUMO
Cofilin is a ubiquitous, actin-binding protein. Only unphosphorylated cofilin binds actin and severs or depolymerizes filamentous actin (F-actin), and the inactive form of cofilin is phosphorylated at Ser 3. We reported recently that cofilin plays a regulatory role in superoxide production and phagocytosis by leukocytes, and in the present study, we investigated the role of cofilin in the chemotaxis of neutrophilic HL-60 cells. IL-8 is a potent, physiological chemokine, and it triggers a rapid, transient increase in F-actin beneath the plasma membrane and rapid dephosphorylation and subsequent rephosphorylation of cofilin. In this study, cofilin phosphorylation was found to be inhibited by S3-R peptide, which consists of a peptide corresponding to part of the phosphorylation site of cofilin and a membrane-permeable arginine polymer. When S3-R peptide was introduced into the neutrophilic cells, their chemotactic activity was enhanced, whereas a control peptide that contained an inverted sequence of the phosphorylation site of cofilin had no enhancing effect. Cofilin small interfering RNA (siRNA) decreased cofilin expression by about half and inhibited chemotaxis. In IL-8-stimulated cells, unphosphorylated cofilin accumulated around F-actin, and colocalization of F-actin and phosphorylated cofilin was observed, but these changes in cofilin localization were less prominent in cofilin siRNA-treated cells. The inhibitors of PI-3K wortmannin and LY294002 inhibited the chemotaxis and suppressed IL-8-evoked dephosphorylation and rephosphorylation of cofilin. These results suggested that unphosphorylated cofilin plays a critical role in leukocyte chemotaxis and that PI-3K is involved in the control of the phosphorylation/dephosphorylation cycle of cofilin.
Assuntos
Fatores de Despolimerização de Actina/fisiologia , Quimiotaxia/imunologia , Interleucina-8/fisiologia , Neutrófilos/imunologia , Fatores de Despolimerização de Actina/efeitos dos fármacos , Actinas/efeitos dos fármacos , Actinas/metabolismo , Androstadienos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Quimiotaxia/efeitos dos fármacos , Cromonas/farmacologia , Células HL-60 , Humanos , Interleucina-8/farmacologia , Morfolinas/farmacologia , Neutrófilos/efeitos dos fármacos , Peptídeos/farmacologia , Fosforilação , RNA Interferente Pequeno/farmacologia , Relação Estrutura-Atividade , WortmaninaRESUMO
Indirubin, a purple vegetable dye, is a traditional Chinese medicine for myelocytic leukaemia. Indirubin inhibits cyclin-dependent protein kinases (CDKs) and is present in human urine and serum. When indirubin was present during the neutrophilic differentiation of human myelocytic leukaemia HL-60 cells, it augmented superoxide production triggered by opsonized zymosan (OZ) by the terminally differentiated HL-60 cells. It also augmented the calcium response to OZ stimulation, and HL-60 cell chemotaxis evoked by interleukin-8 (IL-8, CXCL8) and formylpeptide. In addition, indirubin induced marked IL-8 release by the cells during differentiation and the cells differentiated with indirubin had typical neutrophilic properties, deformed nuclei and granules. Use of stable cloned HL-60 cells that contained a reporter vector for monitoring the activity of the transcription factor PU.1, which acts specifically at the stage of promyelocyte differentiation into neutrophils and monocytes, revealed that indirubin has a potent promoting activity on intracellular PU.1. Indirubin enhanced the expression of typical neutrophil proteins, including granulocyte-colony stimulating factor receptor, the beta2-integrin subunit CD18, the NADPH-oxidase subunit p47phox, and the IL-8 receptor CXCR1, all are controlled by PU.1. Indirubin also inhibited CDK2-dependent phosphorylation of retinoblastoma protein during neutrophilic differentiation. These results suggest that indirubin augments the neutrophilic differentiation of human myelocytic leukaemia HL-60 cells through inhibition of CDK2 and activation of PU.1.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Leucemia Mieloide/patologia , Neutrófilos/efeitos dos fármacos , Antígenos CD18/metabolismo , Diferenciação Celular , Quimiotaxia , Dimetil Sulfóxido/farmacologia , Relação Dose-Resposta a Droga , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células HL-60 , Humanos , Processamento de Imagem Assistida por Computador , Immunoblotting , Indóis/farmacologia , Interleucina-8/metabolismo , Interleucina-8/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Fosforilação , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Receptores de Interleucina-8A/metabolismo , Proteína do Retinoblastoma/metabolismo , Estimulação Química , Superóxidos/metabolismoRESUMO
Bisphenol A (BPA) is a well-known endocrine disruptor. However, little information is available on its immunological effects. To investigate the effect of BPA on leukocyte differentiation, we investigated its action on the neutrophilic differentiation of HL-60 cells induced by dimethylsulfoxide and granulocyte colony-stimulating factor (G-CSF) for 6 days. At low concentrations (10(-10)-10(-8) M), BPA significantly increased the superoxide production by differentiated HL-60 cells stimulated with opsonized zymosan (OZ) by about 20%, and expression of CD18, a component of the OZ-receptor, was increased to a similar extent by 10(-9) M BPA. To investigate the effect of BPA on the activity of PU.1, a transcription factor specific for granulocytic differentiation, we established a stable clone that expressed luciferase as a reporter of PU.1 activity. PU.1 activity increased during the neutrophilic differentiation of HL-60 cells, reaching a peak on day 3 and decreasing thereafter. Nanomolar BPA augmented the PU.1 activity on day 3 by about 60%. On the other hand, tamoxifen, a competitive inhibitor of estrogen receptors, did not suppress the effect of BPA on the differentiation of HL-60 cells. These results suggest that BPA exerts an enhancing effect on the neutrophilic maturation of leukocytes through an estrogen receptor-independent pathway. Long-term exposure to BPA might significantly affect the innate immunity of mammals, even at low doses.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Neutrófilos/fisiologia , Fenóis/farmacologia , Compostos Benzidrílicos , Antígenos CD18/genética , Antígenos CD18/metabolismo , Diferenciação Celular/fisiologia , Dimetil Sulfóxido/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Humanos , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Superóxidos/metabolismo , Tamoxifeno/farmacologia , Transativadores/genética , Transativadores/metabolismo , Zimosan/farmacologiaRESUMO
Triphenyltin (TPT) is an environmental endocrine disruptor and toxic substance, but little information is available on its immunological effects. To assess the effect of TPT on leukocyte differentiation, we investigated its effect on the neutrophilic differentiation of HL-60 cells induced by dimethyl sulfoxide and granulocyte colony-stimulating factor (G-CSF) for 6 days. At a low concentration, 10(-7)M, TPT increased superoxide production by differentiated HL-60 cells stimulated with opsonized zymosan (OZ) by about 45% and increased expression of CD18, a component of the OZ-receptor, by about 90%. Real-time PCR analysis revealed that TPT augmented the expression not only of CD18 but also of components of superoxide-generating NADPH-oxidase, p47phox, 2.7-fold, and p67phox, 2.0-fold, and of granulocyte colony-stimulating factor receptor (G-CSFR), 3.0-fold, whereas various other endocrine disruptors, including parathion, vinclozolin, and bisphenol A, had no such enhancing effects. The results of a DNA macroarray analysis showed that TPT enhanced the expression of G-CSFR and certain other neutrophil functional proteins, including CD14 and myeloid leukemia cell differentiation protein (MCL-1), and that TPT induced a decrease in expression of LC-PTP, leukocyte protein-tyrosine phosphatase, to about half the control level. The TPT-dependent suppression of LC-PTP was confirmed by real-time PCR analysis, and the results of immunoblotting indicated that TPT enhances the expression of myeloid specific tyrosine kinase hck by about 30% at the protein level, and this together with the reduction of LC-PTP may enhance tyrosine phosphorylation, in turn resulting in enhancement of superoxide production. These findings suggest that TPT may have an enhancing effect on the neutrophilic maturation of leukocytes.
Assuntos
Neutrófilos/efeitos dos fármacos , Compostos Orgânicos de Estanho/toxicidade , Antígenos CD18/genética , Antígenos CD18/metabolismo , Diferenciação Celular/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , NADPH Oxidases/genética , Neutrófilos/citologia , Neutrófilos/metabolismo , Fosfoproteínas/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-hck , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Superóxidos/metabolismoRESUMO
We have previously reported that cofilin, an actin-binding protein, plays an important role in phagocyte functions, such as respiratory burst, phagocytosis, and chemotaxis. On the other hand, it was recently found that LIM motif-containing kinase (LIMK) phosphorylates cofilin. In this work, we investigated the roles of LIMK in activated phagocytes. The results of immunostaining showed that in dormant phagocytes the endogenous LIMK1 was diffusely distributed in the cytosol of macrophage-like U937 cells, and when activated by opsonized zymosan (OZ), it was translocated to plasma membranes. Green fluorescence protein (GFP)-conjugated LIMK was expressed in the phagocytes, and the GFP-positive cells were isolated by a fluorescence-activated cell sorter. The isolated wild-type LIMK-overexpressing cells produced superoxide at a rate that was 3.2-fold higher than that of only GFP-expressing control cells, whereas the respiratory burst of dominant negative LIMK1(D460A)-expressing cells decreased to 31% of that of the control cells. Phagocytic activity monitored by using Texas Red-labeled OZ was also decreased in the D460A-expressing cells. By immunoblotting using a specific anti-phosphorylated cofilin antibody, it was revealed that in the OZ-activated wild-type LIMK1-GFP-expressing cells, the phosphorylated cofilin increased by 2.3-fold, and that in the OZ-activated D460A-GFP-expressing cells, the phosphorylated cofilin decreased to 47% of that of only GFP-expressing cells (mock control). Furthermore, in the wild-type LIMK1-expressing cells, OZ-evoked increase in filamentous actin was markedly enhanced, whereas in the dominant negative LIMK1-expressing cells, the total level of F-actin was strongly suppressed. These results suggest that LIMK1 regulates the functions of phagocytes through phosphorylation of cofilin and enhances the formation of filamentous actin.