RESUMO
OBJECTIVE: We compared the in vitro effects of red wine, white wine and ethanol on the cell mediated oxidation of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) by three frequently-used assays. METHODS: LDL and HDL isolated from normolipidemic human serum were incubated with J774.A1 macrophages in DMEM with copper, with or without red wine, white wine or ethanol (equivalent to 0.2 mg ethanol/ml). Lipoprotein oxidation was assessed by conjugated diene formation as measured by changes in absorbance at 234 nm (deltaA234), thiobarbituric-acid-reactive-substance (TBARS) production and trinitrobenzene-sulfonic-acid (TNBS) reactivity. RESULTS: Red wine (0.2 mg ethanol/mL) inhibited LDL oxidation as indicated by an 85.7% decrease in absorbance at 234 nm, a 96.5% decrease in TBARS production and complete prevention of the decrease in TNBS reactivity. White wine and ethanol did not have any significant effect at 0.2 mg/mL. White wine at 1.0 mg ethanol/mL inhibited TBARS production from LDL by 84.1%. Red wine (0.2 mg ethanol/mL) inhibited HDL oxidation as indicated by a 78.9% decrease in deltaA234, an 81.7% decrease in TBARS production and by no change in TNBS reactivity. White wine and ethanol had no effect at 0.2 mg/mL. White wine at 1.0 mg ethanol/mL inhibited TBARS production from HDL by 66.4%. CONCLUSIONS: These results indicate that red wine inhibits the cell mediated oxidation of lipoproteins, that white wine is not as effective as red wine and that the effect of the red wine is not due to its ethanol content.
Assuntos
Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Vinho , Animais , Linhagem Celular , Cobre/química , Etanol/farmacologia , Humanos , Lipoproteínas HDL/química , Lipoproteínas LDL/química , Macrófagos/metabolismo , Masculino , Camundongos , Oxirredução , Espectrofotometria , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Ácido Trinitrobenzenossulfônico/químicaRESUMO
In vitro oxidation of high-density lipoprotein (HDL) diminishes its capacity to mediate cholesterol efflux from J774 macrophages. To investigate the possible role of HDL glycation in the increased atherosclerotic risk in diabetes, we studied the effects of in vitro glycation of HDL on its susceptibility to oxidation and capacity to mediate cholesterol efflux. HDL isolated from normal volunteers was incubated with 25 mmol/L glucose for 70 hours, resulting in 6.1% additional derivatization of apoproteins as determined by trinitrobenzene sulfonic acid (TNBS) reactivity. Unmodified HDL and glycated HDL (glyHDL) were tested for susceptibility to oxidation by incubation with various concentrations of copper and three assays of lipid oxidation. GlyHDL produced 51% to 64% less lipid peroxide than HDL as determined by reaction with xylenol orange (P < .02), indicating decreased susceptibility to oxidation. However, glycation of HDL did not result in significant changes in the formation of conjugated dienes or thiobarbituric acid-reactive substances (TBARS), two other indices of oxidation. To study cholesterol efflux, J774 macrophages were labeled with 3H-cholesterol followed by incubation with the various HDL preparations. HDL and glyHDL had a similar capacity to mediate efflux. The efflux mediated by oxidized HDL (oxHDL) and oxidized glyHDL was reduced to a similar extent compared with the efflux mediated by HDL and glyHDL. These data indicate that in vitro glycation of HDL does not increase its susceptibility to oxidation and does not diminish its capacity to mediate cholesterol efflux.
Assuntos
Colesterol/metabolismo , Glucose/química , Lipoproteínas HDL/química , Lipoproteínas HDL/farmacologia , Humanos , Técnicas In Vitro , Peróxidos Lipídicos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Oxirredução , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
UNLABELLED: Monocytes responding to oxidized low density lipoprotein (LDL) or other antigens may initiate atherogenesis through production of interleukin-1 (IL-1) and additional cytokines. Interleukin-1 is chemotactic for circulating leukocytes, can stimulate growth of fibroblasts or smooth muscle cells, and causes activation of T- and B-lymphocytes. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 42 patients with angiographically verified ischemic heart disease (IHD) and 35 age-matched control subjects without a history of cardiac disease. Rates of proliferation and production of IL-1 beta were measured after peripheral blood mononuclear cells were cultured for 7 days in the presence of mitogens, arterial antigen, lipopolysaccharide, or native and oxidized forms of LDL. RESULTS: In patients with IHD, proliferation in response to arterial antigen was either diminished or unchanged from control values. Peripheral blood mononuclear cells from IHD and control patients had similar levels of proliferation after treatment with different mitogens. Levels of IL-1 beta, produced after stimulation with arterial antigen or lipopolysaccharide, also did not differ for PBMCs obtained from control and IHD patients. For patients with either a stable angina pattern or no history of cardiac disease, PBMC cultured in the presence of native and oxidized forms of LDL released similar amounts of IL-1 beta. In contrast, PBMCs from 4 patients with unstable angina had increased levels of IL-1 beta after culture in the presence of oxidized LDL (group means +/- standard deviation of 1.63 +/- 1.08 pg/mL for 17 control patients, 0.96 +/- 0.23 pg/mL for 4 cases with stable angina, and 4.02 +/- 5.91 pg/mL, for 19 cases with unstable angina). These values reflect a greater than 5-fold increase in variability for IL-1 beta produced on exposure to oxidized LDL for patients with unstable angina relative to control patients. CONCLUSIONS: Effects of in vitro stimulation with mitogens or lipopolysaccharide are similar for PBMC obtained from normal or IHD patients. The response to arterial antigen is also not increased in cells from patients with IHD. However, PBMCs obtained from a subset of patients with unstable angina produce greater levels of IL-1 beta after treatment with oxidized, but not native, LDL.
Assuntos
Interleucina-1/biossíntese , Lipopolissacarídeos/farmacologia , Lipoproteínas LDL/farmacologia , Mitógenos/farmacologia , Isquemia Miocárdica/metabolismo , Túnica Íntima/imunologia , Adulto , Antígenos/farmacologia , Células Cultivadas , Feminino , Humanos , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Isquemia Miocárdica/imunologia , OxirreduçãoRESUMO
In vitro studies suggest that oxidized low density lipoprotein inhibits fibrinolysis by stimulating the production of plasminogen activator inhibitor -1 (PAI). We assessed the effects of dietary antioxidant vitamins for four weeks on three indices of copper mediated oxidation of very low and low density lipoproteins (VLDL+LDL) and plasma fibrinolytic activities in 15 male subjects with central obesity, a condition associated with increased PAI activity. Vitamin administration resulted in a decrease in production of thiobarbituric acid reactive substances from 29.3 +/- 3.9 to 13.6 +/- 3.5 nmoles/mg VLDL + LDL protein (mean +/- SE, p <0.003), an increase in the lag phase of conjugated diene formation from 94.8 +/- 5.5 to 225.0 +/- 31.9 min (p <0.001) and an increase in reactivity of lysine residues from 73.6% +/- 4.8% to 86.8% +/- 3.6% (p <0.034) demonstrating a reduction in the susceptibility of the lipoproteins to oxidation. However, antioxidant vitamins had no effect on plasma PAI activity, PAI antigen, tissue-type plasminogen activator activity and antigen, fibrinogen and fibrin degradation products. These results do not support the hypothesis that lipoprotein oxidation is a significant cause of impaired fibrinolysis in men with central obesity.
Assuntos
Antioxidantes/farmacologia , Fibrinólise/efeitos dos fármacos , Obesidade/fisiopatologia , Vitaminas/farmacologia , Ácido Ascórbico/sangue , Glicemia/análise , Humanos , Lipoproteínas LDL/sangue , Lipoproteínas VLDL/sangue , Masculino , Inibidor 1 de Ativador de Plasminogênio/sangue , Ativador de Plasminogênio Tecidual/metabolismo , Ácido Trinitrobenzenossulfônico/análiseRESUMO
The purpose of our study was to examine the relationship between insulin resistance and blood pressure during pregnancy and to determine to what extent insulin resistance is related to the subsequent development of pregnancy-induced hypertension. The study population consisted of 292 women who had serum insulin, glucose and insulin-glucose ratios determined at 26-28 weeks gestation in a fasting state and 1 hr after a 50-g oral glucose challenge. These were compared with blood pressures at 26-28 weeks gestation and in the late third trimester. A statistically significant correlation exists overall between (1) blood pressure at 26-28 weeks gestation and both fasting insulin and insulin-glucose ratios, as well as (2) systolic blood pressure at term and fasting insulin levels. However, when controlled for confounding variables including body mass index, race and age, no statistically significant relationship remained. The metabolic variables in patients with pregnancy-induced hypertension were not statistically different from the normotensive patients. In conclusion, this study demonstrates that insulin resistance and hyperinsulinemia are not major determinants of blood pressure during pregnancy.
Assuntos
Pressão Sanguínea/fisiologia , Hiperinsulinismo/fisiopatologia , Resistência à Insulina/fisiologia , Complicações na Gravidez/fisiopatologia , Adulto , Feminino , Humanos , Hipertensão/etiologia , Hipertensão/fisiopatologia , Gravidez , Complicações Cardiovasculares na Gravidez/fisiopatologia , Segundo Trimestre da Gravidez , Estudos ProspectivosRESUMO
Copper mediated oxidative modification of high density lipoprotein (HDL) diminishes its capacity to promote cholesterol efflux from cells in culture. In the present study, HDL was isolated from eight subjects before and after a 10 day administration of the antioxidant vitamins C and E. After incubation HDL (1.25 mg protein/ml) with 10 microM copper for 0-4 h or with 0-20 microM copper for 4 h, thiobarbituric acid reactive substances (TBARS) production was significantly decreased following vitamin administration suggesting that the vitamins decreased the susceptibility of HDL to oxidation. However, two other assays of lipoprotein oxidation, trinitrobenzene sulfonic acid reactivity and conjugated diene formation, did not show a consistent effect of vitamin administration. To study cholesterol efflux, J774 macrophages were labeled with 3H cholesterol (0.1 microCi/ml, 50 micrograms/ml) and incubated with HDL or oxidized HDL (100 micrograms protein/ml) for 24 h. HDL isolated before vitamins and oxidized in vitro was 39% less effective in mediating efflux compared to unmodified HDL, while HDL isolated after vitamins and oxidized was 22% less effective (before vs. after vitamins, P < 0.015). HDL oxidation determined by measuring TBARS production correlated with decreased cholesterol efflux (r = 0.37, P < 0.050). These data suggest that oxidation of HDL interferes with its role in reverse cholesterol transport and that antioxidant vitamins have a protective effect.
Assuntos
Ácido Ascórbico/administração & dosagem , Colesterol/metabolismo , Cobre/farmacologia , Lipoproteínas HDL/metabolismo , Vitamina E/administração & dosagem , Adulto , Animais , Células Cultivadas , Doença das Coronárias/sangue , Doença das Coronárias/etiologia , Doença das Coronárias/prevenção & controle , Feminino , Alimentos Fortificados , Humanos , Lipoproteínas HDL/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Oxirredução/efeitos dos fármacos , Peritônio/citologia , Valores de Referência , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Ácido Trinitrobenzenossulfônico/metabolismoRESUMO
Oxidative modification of high density lipoproteins (HDL) may alter their capacity to mediate cellular cholesterol efflux. We studied the kinetics of copper-mediated oxidation of HDL and cholesterol efflux mediated by unmodified and oxidized HDL (oxHDL). Oxidation was measured by increases in absorbance at 234 nm (delta A234), production of thiobarbituric acid reactive substances (TBARS) and loss of trinitrobenzene sulfonic acid reactivity. Oxidation was dependent on copper concentration and showed a lag phase and propagation phase. Efflux of cholesterol from J774 macrophages measured by appearance of cellular [3H]cholesterol in the medium was lower by 16% after 4 h and 36% after 24 h with oxHDL compared to HDL. OxHDL-mediated efflux was also lower by 27% to 37% at lipoprotein concentrations of 10 to 200 micrograms protein/ml. Cholesterol efflux correlated negatively with TBARS production (r = -0.97, P < 0.003) and delta A234 (r = -0.77, P < 0.080). There was no difference in efflux mediated by apoproteins prepared from HDL and oxHDL. Efflux measured by change in cholesterol mass in medium was 78% lower with oxHDL. Inhibition of oxidation with butylated hydroxytoluene maintained the capacity of HDL to stimulate efflux. These results suggest that oxidation of HDL may impair its protective role against atherosclerosis.
Assuntos
Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , Animais , Arteriosclerose/metabolismo , Linhagem Celular , Cobre/farmacologia , Lipoproteínas HDL/química , Camundongos , Oxirredução , Espectrofotometria Ultravioleta , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Ácido Trinitrobenzenossulfônico/análiseRESUMO
Hyperinsulinemia has been implicated as an independent risk factor for atherosclerosis. We measured the effect of insulin and related hormones on the oxidation of low density lipoproteins (LDL) and superoxide anion production by peripheral blood mononuclear cells (MC). LDL oxidation was measured by the production of thiobarbituric acid reactive substances (TBARS). Insulin and IGF-I at 10(-7) M caused a 33% and 48% increase in TBARS production, respectively. At 10(-6) M the corresponding values were 63% and 67%. Proinsulin and IGF-II at 10(-6) M had no effect. Glucose caused a concentration dependent (up to 10 mM) stimulation of LDL oxidation reaching 85% and 77% at insulin concentrations of 10(-7) M and 10(-6) M, respectively. The stimulatory effect of insulin was confirmed by measurements of other indices of LDL oxidation, i.e. absorbance at 234 nm, trinitrobenzene sulfonic acid reactivity and electrophoretic mobility. Insulin-stimulated LDL oxidation was inhibited by superoxide dismutase (SOD), but insulin had no effect on MC superoxide production. MC were isolated from five subjects before and after a 5 h hyperinsulinemic, euglycemic clamp. Insulin infusion had no effect on TBARS or superoxide production by MC. Our in vitro experiments suggest that high levels of insulin and IGF-I stimulate MC-mediated oxidation of LDL, an effect that is potentially atherogenic.
Assuntos
Fator de Crescimento Insulin-Like I/fisiologia , Insulina/fisiologia , Leucócitos Mononucleares/fisiologia , Lipoproteínas LDL/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 1/fisiopatologia , Humanos , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Lipoproteínas LDL/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Substâncias Reativas com Ácido Tiobarbitúrico/análiseRESUMO
The oxidative modification of lipoproteins has been implicated in atherogenesis, suggesting a protective role of circulating antioxidants. Vitamin C (ascorbic acid, 1 g/day) and vitamin E (dl alpha-tocopheryl acetate, 800 IU/day) were administered to healthy female and male volunteers. Lipoproteins with density < 1.063 g/mL were isolated from serum before and after vitamin supplementation and incubated with copper (Cu) or mononuclear cells (MC) plus Cu. Administration of vitamins C and E together to 4 subjects for 10 days resulted in a 57% (range 40-72%) decrease in Cu-catalyzed production of thiobarbituric acid reactive substances (TBARS) under the following conditions of assay: incubation times of 0-8 hours, Cu concentrations of 0-10 microM lipoprotein protein concentrations of 0.1-0.5 mg/mL. Decreases in other parameters of lipoprotein oxidation, i.e,, electrophoretic mobility, production of conjugated dienes and modification of amino groups, were also observed. Vitamin E administration alone produced a 52% inhibition and vitamin C alone a 15% inhibition of TBARS formation. Vitamins C and E supplementation resulted in a 78% decrease in the susceptibility of lipoproteins to MC-mediated oxidation. There was a strong inverse correlation (r = -0.64, p < 0.0007) between vitamin E levels in the lipoproteins and TBARS production in samples from 12 subjects administered vitamins C and E. In 3 individuals vitamin E levels remained low and in 2 of these subjects there was no effect of vitamins C and E administration on TBARS production. These results suggest a protective role of antioxidant vitamins and significant individual variability in response.
Assuntos
Ácido Ascórbico/farmacologia , Dieta , Peroxidação de Lipídeos , Lipoproteínas/sangue , Vitamina E/farmacologia , Adulto , Ácido Ascórbico/administração & dosagem , Cobre/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxirredução , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Vitamina E/administração & dosagemRESUMO
The antioxidant activities of 17-beta-estradiol (E2) and other steroid hormones were studied by determining their effect on copper-catalyzed (cell-free) and mononuclear cell-mediated oxidation of low-density lipoproteins (LDL), as measured by the production of thiobarbituric acid-reactive substances (TBARS). The oxidation of LDL increased linearly with copper concentrations ranging from 0 to 10 mumol/L. E2 at a concentration of 1 mumol/L inhibited LDL oxidation by 37% to 62% at the various concentrations of copper. In a time-course study, E2 at 1 mumol/L delayed the onset of LDL oxidation in the presence of 5 mumol/L copper. E2 (1 mumol/L) inhibited TBARS production catalyzed by 5 mumol/L copper by 54%, compared with 60% inhibition by 1 mumol/L butylated hydroxytoluene (BHT), a known inhibitor of lipid peroxidation. Estriol at 5 mumol/L decreased LDL oxidation by 49%. Dehydroepiandrosterone (DHEA), testosterone, and estrone had no significant effects. E2 was also an effective inhibitor of mononuclear cell (MNC)-mediated oxidation of LDL, but had no effect on superoxide production by these cells. The onset of TBARS formation from cell-mediated LDL oxidation was also delayed by incubation with 1 mumol/L E2. The results indicate that estrogen may protect against atherosclerosis by inhibiting lipoprotein oxidation.
Assuntos
Estradiol/farmacologia , Lipoproteínas LDL/metabolismo , Antioxidantes/farmacologia , Hidroxitolueno Butilado/farmacologia , Estradiol/sangue , Estriol/farmacologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Oxirredução/efeitos dos fármacos , Superóxidos/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Fatores de TempoRESUMO
Hyperlipidemia may play a role in the progression of diabetic and other renal diseases. Low density lipoprotein (LDL) and other proteins including extracellular matrix components undergo nonenzymatic glycation in vivo. We examined the effects of glycation of LDL as occurs in diabetes (4 to 8%) on binding and uptake by mesangial cells and their proliferation. The glycation of LDL (g-LDL) significantly decreased its binding and uptake by mesangial cells by 15 to 20%, indicating that glycated LDL binds to the LDL receptor, but with lower affinity than LDL. Both LDL and g-LDL modestly stimulated [3H] thymidine incorporation into mesangial cells at 5 to 10 micrograms/ml. Native, oxidized (Ox-LDL) and glycated LDL all bound to the extracellular matrix generated by rat mesangial cells in culture. The binding of LDL, Ox-LDL and g-LDL to mesangial matrix was two to four times higher than to mesangial cells. Binding of LDL and g-LDL was significantly higher to glycolaldehyde modified matrix, which serves as an in vitro model for nonenzymatic glycation end-product cross-linking of matrix which occurs in long-standing diabetes. Based on these findings, we propose that glycation of LDL decreases its binding and uptake by the LDL receptor of mesangial cells and may slow its catabolism. Furthermore, LDL bound to extracellular mesangial matrix can undergo oxidation and generate cytotoxic LDL components. This process may be further enhanced by advanced glycation of the mesangial matrix in diabetes, contributing to glomerular pathology.
Assuntos
Mesângio Glomerular/metabolismo , Lipoproteínas LDL/metabolismo , Animais , Divisão Celular , Matriz Extracelular/metabolismo , Mesângio Glomerular/citologia , Glucose/metabolismo , Produtos Finais de Glicação Avançada , Glicosilação , Técnicas In Vitro , Masculino , Oxirredução , Ratos , Ratos EndogâmicosRESUMO
Hyperlipidemia may contribute to the pathogenesis of glomerular sclerosis. We therefore compared binding and uptake of native LDL and oxidized LDL (Ox-LDL) to cultured mesangial cells (MC) and the resulting effects on prostaglandin generation and cell proliferation. Ox-LDL, prepared from native LDL by incubation with copper, was bound to MC in a concentration dependent manner with a four- to fivefold increase in binding over LDL. In competition binding experiments Ox-LDL competed to 90% with LDL for binding sites, but LDL only displaced Ox-LDL to 15%. Furthermore polyinosinic acid, which blocks binding of Ox-LDL to macrophages, inhibited binding of Ox-LDL but not that of LDL to MC. Mesangial cells also preferentially took up Ox-LDL over LDL, and Ox-LDL resulted in higher [14C] oleate incorporation into cholesteryl esters than LDL, findings consistent with different handling of Ox-LDL and LDL by MC. LDL slightly stimulated mesangial cell proliferation at low concentration (10 to 50 micrograms/ml of LDL) returning to control levels at 100 and 250 micrograms/ml. In contrast Ox-LDL inhibited cell proliferation in a concentration-dependent manner, starting at concentrations as low as 10 to 25 micrograms/ml of Ox-LDL. Direct observations of mesangial cells by phase contrast microscopy confirmed the cytotoxic effects of Ox-LDL. Addition of Ox-LDL to mesangial cells resulted in a concentration-dependent increase in PGE2 synthesis within one hour, while at this time point LDL had no significant effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Mesângio Glomerular/metabolismo , Glomérulos Renais/metabolismo , Lipoproteínas LDL/metabolismo , Animais , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Dinoprostona/metabolismo , Eletroforese em Gel de Ágar , Mesângio Glomerular/citologia , Lipoproteínas LDL/farmacocinética , Masculino , Oxirredução , Ratos , Ratos EndogâmicosRESUMO
The objective of this study was to establish conditions whereby apoB secreted from HepG2 cells could be regulated over a wide range, and to determine whether changes of output were correlated with the level of apoB mRNA. The presence of oleate (complexed to 3% albumin at a molar ratio of 1.7:1) resulted in a 3.5-fold stimulation of apoB secretion that was apparent after only 3 h. Insulin halved the rate of apoB output and the inhibition was detectable within the physiological insulin range, but was not apparent until 12-16 h. Albumin in the culture medium had a dose-dependent inhibitory effect on apoB production. Overall, apoB secretion from HepG2 cells was modulated over a 7-fold range. However, when apoB mRNA was assayed by slot-blot hybridization, no change was detectable under any of the conditions that modulated apoB output. Quantitative solution hybridization was used to confirm that oleate did not affect the level of apoB mRNA. Kinetic analysis of the decay of [3H]uridine-labeled apoB mRNA showed that the half-life of apoB mRNA was 16 h. We conclude from these studies that the apoB gene is constitutively expressed in HepG2 cells and that the mechanism of acute regulation of apoB production by these cells must involve co- or post-translational processes.
Assuntos
Albuminas/farmacologia , Apolipoproteínas B/biossíntese , Regulação da Expressão Gênica , Insulina/farmacologia , Ácidos Oleicos/farmacologia , RNA Mensageiro/metabolismo , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Northern Blotting , Sondas de DNA , Ensaio de Imunoadsorção Enzimática , Meia-Vida , Humanos , Ácido Oleico , Células Tumorais CultivadasRESUMO
Hyperlipidemia may contribute to the pathogenesis of glomerular sclerosis. We therefore studied binding and uptake of low density lipoprotein (LDL) by cultured rat mesangial cells. In addition effects of LDL on PGE2 synthesis and cell proliferation were determined. At 4 degrees C mesangial cells bound [125I] LDL in a time- and concentration-dependent manner with half-maximal binding observed at 5 micrograms/ml of LDL protein. Binding was blocked by excess unlabeled LDL and by heparin. Uptake (binding plus internalization) of LDL at 37 degrees C markedly exceeded binding at 4 degrees C, continued to increase even with longer periods of incubation, and showed no saturability, consistent with uptake of LDL by mesangial cells. Further evidence for LDL uptake by mesangial cells was obtained by use of the fluorescent probe 1,1'-dioactadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate-labeled LDL (Dil-LDL). Incubation of mesangial cells with Dil-LDL at 37 degrees C showed positive fluorescence for all mesangial cells, indicating uptake of the Dil-LDL. LDL had a biphasic effect on mesangial cell proliferation as determined by [3H] thymidine incorporation. LDL at 10 micrograms/ml enhanced [3H] thymidine uptake modestly, but significantly, whereas a progressive and marked inhibition occurred at LDL concentration from 100 to 500 micrograms/ml. While LDL at 10 and 100 micrograms/ml significantly stimulated PGE2 production, inhibition of PGE2 by meclofenamate did not influence the effects of LDL on [3H] thymidine incorporation. We conclude that mesangial cells show specific binding and uptake of LDL and that high concentrations of LDL markedly decrease mesangial cell proliferation. These findings may pertain to the pathogenesis of glomerular lesions in hyperlipidemia of renal disease.
Assuntos
Mesângio Glomerular/metabolismo , Receptores de LDL/metabolismo , Animais , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , Replicação do DNA/efeitos dos fármacos , Dinoprostona/biossíntese , Mesângio Glomerular/citologia , Mesângio Glomerular/efeitos dos fármacos , Humanos , Cinética , Lipoproteínas LDL/sangue , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , Masculino , Ratos , Ratos Endogâmicos , Timidina/metabolismoRESUMO
The receptor-mediated uptake of very low density lipoprotein (VLDL) remnants by the rat liver was studied. Livers were perfused with native 125I-VLDL remnants, radiolabeled apo E-deficient remnants, and radiolabeled remnants that contained reductively methylated apo B and unmodified apo E. The specific uptake of the apo E-deficient remnants was 20% of that for the native remnants, whereas the specific uptake of the remnants containing unreactive apo B was 78% of the control value. This suggests that the apo E of VLDL remnants is the principal ligand for binding to the receptor, and in the absence of apo E, apo B may participate in binding. This conclusion is supported by the finding that dimyristoyl phosphatidylcholine (DMPC)- apo E complexes were effective in competing for the hepatic uptake of 125I-VLDL remnants. The intracellular distribution of radioactivity was analyzed by Percoll density gradient centrifugation. At five minutes after perfusion, radioactivity was associated with the plasma membrane and lysosomal fractions, and at 30 minutes most of the radioactivity was associated with the lysosomal fraction. Binding and internalization of VLDL remnants was also directly visualized by electron microscopy. Internalization proceeded by coated pit-coated vesicle formation with subsequent delivery to lysosomes. Our findings demonstrate that the apo E of VLDL remnants mediates binding to the hepatic receptor and that the internalization and degradation of VLDL remnants is by a similar pathway to that previously described for LDL.
Assuntos
Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Receptores de LDL/metabolismo , Animais , Transporte Biológico , Radioisótopos do Iodo , Cinética , Lipoproteínas VLDL/sangue , Fígado/ultraestrutura , Masculino , Microscopia Eletrônica , Peso Molecular , Perfusão , Ratos , Ratos EndogâmicosRESUMO
We have used a preparation of rat liver plasma membranes to study the binding of rat apolipoprotein E-deficient HDL to rat liver. The membranes were found to bind HDL by a saturable process that was competed for by excess unlabeled HDL. The binding was temperature-dependent and was 85% receptor-mediated when incubated at 4, 22 and 37 degrees C. The affinity of the binding site for the HDL was consistent at all temperatures, while the maximum binding capacity increased at higher temperatures. The specific binding of HDL to the membranes did not require calcium and was independent of the concentration of NaCl in the media. The effect of varying the pH of the media on HDL binding was small, being 30% higher at pH 6.5 than at pH 9.0. Both rat HDL and human HDL3 were found to compete for the binding of rat HDL to the membranes, whereas rat VLDL remnants and human LDL did not compete. At 4 degrees C, complexes of dimyristoylphosphatidylcholine (DMPC) and apolipoproteins A-I, A-IV and the C apolipoproteins, but not apolipoprotein E, competed for HDL binding to the membranes. At 22 and 37 degrees C, all DMPC-apolipoprotein complexes competed to a similar extent, DMPC vesicles that contained no protein did not compete for the binding of HDL. These results suggest that the rat liver possesses a specific receptor for apolipoprotein E-deficient HDL that recognizes apolipoproteins A-I, A-IV and the C apolipoproteins as ligands.
Assuntos
Proteínas de Transporte , Lipoproteínas HDL/metabolismo , Fígado/metabolismo , Proteínas de Ligação a RNA , Receptores de Superfície Celular/metabolismo , Receptores de Lipoproteínas , Animais , Apolipoproteínas E , Ligação Competitiva , Membrana Celular/metabolismo , Humanos , Técnicas In Vitro , Masculino , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/efeitos dos fármacos , Temperatura , Fatores de TempoAssuntos
Proteínas de Transporte , Lipoproteínas HDL/metabolismo , Fígado/metabolismo , Proteínas de Ligação a RNA , Receptores de Superfície Celular/metabolismo , Receptores de Lipoproteínas , Animais , Membrana Celular/metabolismo , Cinética , Masculino , Peso Molecular , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/isolamento & purificaçãoRESUMO
The uptake of the 125I-labeled apolipoprotein and 3H-labeled cholesteryl ester components of rat apolipoprotein E-deficient HDL by the perfused liver was studied. The uptake of the cholesteryl ester moiety was 4-fold higher than that of apolipoprotein. The concentration-dependent uptake of labeled protein was saturable and competed for by an excess of unlabeled HDL. The uptake of cholesteryl ester was not saturable over the concentration range studied. In the presence of a 50-fold excess of unlabeled HDL, the uptake of both radiolabeled components was decreased by over 75%, indicating that three-quarters of the hepatic uptake of HDL is by a receptor-mediated process. After 15 min of perfusion, 37% of the apolipoprotein radioactivity that was initially bound at 5 min was released into the perfusate as a more dense particle. After 5, 15, 30 and 60 min of perfusion the subcellular distribution of the apolipoprotein and cholesteryl ester components was analyzed by Percoll density gradient centrifugation. Over the 60 min period, there appeared to be transfer of radioactivity from the plasma membrane fraction to the lysosomal fraction. However, the internalization and degradation of cholesteryl ester was more rapid than that of the apolipoprotein. Our findings indicate that there is preferential uptake of HDL cholesteryl ester relative to protein by the liver and that the internalization of these components may occur independently.
Assuntos
Apolipoproteínas A/metabolismo , HDL-Colesterol/metabolismo , Fígado/metabolismo , Animais , Apolipoproteínas E/deficiência , Transporte Biológico , Humanos , Cinética , Lipoproteínas HDL/metabolismo , Masculino , Perfusão , Ratos , Ratos EndogâmicosRESUMO
Apolipoprotein A-IV was isolated from the d less than 1.21 g/ml fraction of rat serum by gel filtration followed by heparin-Sepharose affinity chromatography; this method also facilitated the preparation of apolipoprotein A-I and apolipoprotein E. The apolipoprotein A-IV preparation was characterized by SDS-gel electrophoresis, isoelectric focusing, amino acid analysis and immunodiffusion. The lipid-binding properties of this protein were studied. Apolipoprotein A-IV associated with dimyristoylphosphatidylcholine (DMPC) to form recombinants which contained two molecules of apolipoprotein A-IV and had a lipid/protein molar ratio of 110. The density of the DMPC/apolipoprotein A-IV particles was determined to be 1.08 g/ml and the particles were visualized by electron microscopy as discs which were 5.8 nm thick and 18.0 nm in diameter. The stability of the DMPC/apolipoprotein A-IV recombinants, as determined by resistance to denaturation, was comparable to the stability of DMPC/apolipoprotein A-I complexes. However, by competition studies it was found that apolipoprotein A-I competed for the binding to DMPC more effectively than did apolipoprotein A-IV. It is concluded that, while rat apolipoprotein A-IV resembles other apolipoproteins in its lipid-binding characteristics, it may be displaced from lipid complexes by apolipoprotein A-I.
Assuntos
Apolipoproteínas A/metabolismo , Metabolismo dos Lipídeos , Aminoácidos/sangue , Animais , Apolipoproteína A-I , Apolipoproteínas A/sangue , Ligação Competitiva , Centrifugação com Gradiente de Concentração , Cromatografia em Gel , Dicroísmo Circular , Eletroforese , Heparina , Imunodifusão , Focalização Isoelétrica , Ligação Proteica , RatosRESUMO
The responsiveness of low density lipoprotein (LDL) binding and uptake was measured in a human hepatoblastoma cell line, Hep G2. These cells exhibited both saturable, high-affinity and nonsaturable low-affinity components similar to that described for LDL binding in other mammalian cells. In addition, receptor-mediated uptake of [125I]LDL was dependent on the presence of Ca++ and required intact lysine residues in LDL as evidenced by abolition of binding following reductive methylation of LDL. The receptor activity was rapidly responsive to changes made in LDL concentrations in the medium, up-regulating by 200% in the absence of LDL, and down-regulating by 56% in the presence of LDL, both changes occurring within 2 hr. Cholesterol synthesis was estimated by measuring 3-hydroxymethylglutaryl coenzyme A reductase activity. The activity of this enzyme was also found to increase rapidly in the absence of LDL and decrease rapidly in the presence of LDL in the media. The human cell line Hep G2 possesses an active and responsive receptor for LDL and provides a useful model for the study of lipoprotein uptake and metabolism in intact human liver.