RESUMO
Diabetes is an established risk factor for reinfarction and cardiac death in postinfarction patients. Since the underlying mechanism of diabetes-related risk is not fully understood we aimed to evaluate the association between lipids, thrombogenic factors and diabetes in postinfarction patients. The study population consisted of 1,045 postinfarction patients (846 non-diabetic, 125 non-insulin- and 74 insulin-requiring diabetics) with the following blood tests performed 2 months after an index myocardial infarction: lipoprotein (a), apolipoprotein-B, apolipoprotein-A, cholesterol, HDL cholesterol, triglycerides, insulin, von Willebrand factor (vWF), fibrinogen, factor VII, D-dimer, and plasminogen activator inhibitor (PAI-1). After adjustment for relevant clinical covariates, non-insulin-requiring diabetes was significantly (p < 0.05) associated with elevated levels of (odd ratios per 1 log unit increase in parenthesis) vWF (1.74) and PAI-1 (1.42) whereas insulin requiring diabetes was associated with even more elevated levels of vWF (4.68), but not with increased levels of PAI-1. No significant differences in lipid levels were observed among three groups. In conclusion, increased level of von Willebrand factor is significantly and independently associated with diabetes in postinfarction patients, suggesting that endothelial damage is the primary mechanisms contributing to an increased occurrence of vascular and cardiac events in diabetic postinfarction patients.
Assuntos
Diabetes Mellitus/sangue , Infarto do Miocárdio/sangue , Fator de von Willebrand/análise , Adulto , Idoso , Glicemia/análise , Proteínas Sanguíneas/análise , Convalescença , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Endotélio Vascular/patologia , Feminino , Humanos , Insulina/sangue , Lipídeos/sangue , Lipoproteínas/sangue , Masculino , Pessoa de Meia-Idade , Análise Multivariada , New York/epidemiologia , Razão de Chances , Inibidor 1 de Ativador de Plasminogênio/análise , Fatores de RiscoRESUMO
Lipoprotein and apolipoprotein changes were evaluated in 10-week-old Zucker diabetic fatty (ZDF) male rats following 12 weeks of insulin treatment, which normalized blood glucose and maintained weight gaining characteristic of nondiabetic Zucker fatty rats. Compared with untreated ZDF rats (saline-injected), insulin treatment resulted in increased very-low-density lipoprotein (VLDL; d < 1.006 g/mL) and decreased alpha lipoprotein on agarose gel electrophoresis. These findings were consistent with an observed increase in VLDL triglyceride and cholesterol, and decreased high-density lipoprotein (HDL) cholesterol with insulin treatment in isolated lipoproteins. B100 levels were unchanged by insulin treatment, but B48 levels were significantly increased in the VLDL fraction. Insulin treatment depressed apolipoprotein (apo) A-I levels in HDL, but had little effect on total apo E, apo A-IV, or apo C, although apo C was redistributed to the VLDL fraction. These results suggest that insulin treatment of ZDF rats normalizes hyperglycemia and prevents age-related changes in lipoprotein parameters associated with development of insulinopenic diabetes. Insulin therapy in ZDF rats thereby sustains the hyperlipidemic lipoprotein pattern associated with hyperinsulinemia and obesity.
Assuntos
Hipertrigliceridemia/complicações , Insulina/administração & dosagem , Obesidade/complicações , Animais , Glicemia/análise , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Hiperinsulinismo/sangue , Hiperinsulinismo/complicações , Hipertrigliceridemia/sangue , Lipoproteínas/sangue , Lipoproteínas/classificação , Lipoproteínas/isolamento & purificação , Masculino , Fenótipo , Ratos , Ratos ZuckerRESUMO
Cigarette smoking is linked to increased cardiac morbidity and mortality, and has been shown to affect both lipid profiles and thrombotic factors in healthy subjects. However, the influence of smoking on the atherothrombotic environment has not been studied in a large population of patients after acute myocardial infarction (AMI). Blood samples and medical history, including smoking status, were obtained from 1,045 patients at a 2-month visit after AMI. Smokers were asked to refrain 24 hours before the visit, but not all complied. Measurements included total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, apolipoprotein-B, apolipoprotein-A, triglycerides, factor VII, factor VIIa, von Willebrand factor, D-dimer, and plasminogen activator inhibitor. There were 247 current, 443 past, and 349 nonsmokers. After adjustment for clinical variables, current smokers had higher levels of total cholesterol and apolipoprotein-B than past and nonsmokers (p <0.01). High-density lipoprotein cholesterol and apolipoprotein-A levels were similar between groups. Fibrinogen was elevated in current (p = 0.001) and past (p = 0.029) smokers, compared with nonsmokers. Smokers who smoked within 24 hours of blood sampling had higher apolipoprotein-B (p = 0.005), total cholesterol (p = 0.001), and fibrinogen (p = 0.015) levels than those who refrained from smoking. In conclusion, postinfarction patients, who historically have higher levels of atherogenic lipids than healthy subjects, have increased levels of these lipids attributed to active smoking. After smoking cessation, lipid profiles approach nonsmoker levels, but fibrinogen remains elevated. Smoking within 24 hours of blood sampling was associated with further adverse prothrombotic and lipogenic effects.
Assuntos
Lipídeos/sangue , Infarto do Miocárdio/sangue , Fumar/efeitos adversos , Idoso , Apolipoproteínas B/sangue , Colesterol/sangue , Feminino , Fibrinogênio/análise , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/epidemiologia , Fumar/epidemiologia , Fatores de TempoRESUMO
The current study assessed in vivo the effect of insulin on triglyceride-rich lipoprotein (TRL) production by rat liver. Hepatic triglyceride and apolipoprotein B (apoB) production were measured in anesthetized, fasted rats injected intravenously with Triton WR-1339 (400 mg/kg). After intravascular catabolism was blocked by detergent treatment, glucose (500 mg/kg) was injected to elicit insulin secretion, and serum triglyceride and apoB accumulation were monitored over the next 3 h. In glucose-injected rats, triglyceride secretion averaged 22.5 +/- 2.1 microg.ml(-1).min(-1), which was significantly less by 30% than that observed in saline-injected rats, which averaged 32.1 +/- 1.4 microg.ml(-1).min(-1). ApoB secretion was also significantly reduced by 66% in glucose-injected rats. ApoB immunoblotting indicated that both B100 and B48 production were significantly reduced after glucose injection. Results support the conclusion that insulin acts in vivo to suppress hepatic very low density lipoprotein (VLDL) triglyceride and apoB secretion and strengthen the concept of a regulatory role for insulin in VLDL metabolism postprandially.
Assuntos
Apolipoproteínas B/biossíntese , Glucose/farmacologia , Insulina/metabolismo , Lipoproteínas/biossíntese , Fígado/metabolismo , Triglicerídeos/biossíntese , Animais , Apolipoproteínas B/metabolismo , Glicemia/metabolismo , Células Cultivadas , Dexametasona/farmacologia , Jejum , Glucagon/farmacologia , Glucocorticoides/farmacologia , Técnica Clamp de Glucose , Insulina/farmacologia , Secreção de Insulina , Cinética , Lipoproteínas/sangue , Fígado/efeitos dos fármacos , Masculino , Ácido Oleico/farmacologia , Ratos , Ratos Sprague-Dawley , Triglicerídeos/sangueRESUMO
We recently demonstrated that expression of BHMT in McArdle RH-7777 (McA-BHMT) cells increases apo B mRNA abundance, leading to parallel increases in apo B secretion. The ratio of unedited to edited apo B mRNA was unchanged by BHMT expression. Based on the observation that secretion of B48 is increased relative to B100 in McA-BHMT cells, current studies now include comparison of B48 and B100 synthesis and intracellular degradation. Minor differences in co- and posttranslational degradation were unable to account for relative increase in B48 secretion, and the disappearance kinetics of B48 were similar in McA-BHMT and control cells. Consistent with the increase in endogenous apo B mRNA in McA-BHMT cells, B48 synthesis is increased significantly. In contrast, synthesis of B100 was not significantly increased. We conclude that B48 is preferentially translated compared to B100 when endogenous apo B mRNA is increased.
Assuntos
Apolipoproteínas B/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Animais , Apolipoproteína B-100 , Apolipoproteína B-48 , Betaína-Homocisteína S-Metiltransferase , Linhagem Celular , Metiltransferases/metabolismo , RatosRESUMO
Thrombosis contributes to recurrent coronary events in patients after acute myocardial infarction (AMI), but prognostic significance of thrombogenic factors by gender is unknown. This study aimed to determine gender-related differences in the prognostic significance of thrombogenic factors for predicting cardiac events (nonfatal reinfarction or cardiac death) in postinfarction patients. Blood levels of the following factors were measured 2 months after AMI in 791 men and 254 women: fibrinogen, von Willebrand factor, factor VII and VIIa, plasminogen activator inhibitor, D-dimer, cholesterol, apolipoprotein A-1, apolipoprotein B, lipoprotein(a), triglycerides, and high-density lipoprotein cholesterol. After adjustment for clinical covariates, levels of apolipoprotein A, high-density lipoprotein cholesterol, fibrinogen, and factor VIIa were significantly higher in postinfarction women than men. During a mean 26-month follow-up, there were 67 cardiac events (8.5%) in men and 14 (5.5%) in women (p = 0.11). In the multivariate Cox model, elevated levels of factor VIIa were a significant predictor of cardiac events in women (p = 0.022) but not in men (p = 0.80), with significant gender-related effect (hazard ratio 2.80 vs 0.92, respectively; p <0.05). D-dimer had prognostic value in men (p = 0. 006) but not in women (p = 0.36), although the difference between hazard ratios for men and women was not significant (2.35 vs 1.58, respectively; p = 0.49). In conclusion, elevated levels of factor VIIa are associated with an increased risk of recurrent cardiac events in postinfarction women, but not in men. D-dimer is more predictive for cardiac events in postinfarction men than women. These observations indicate possible gender-related differences in the pathophysiologic mechanisms of recurrent cardiac events.
Assuntos
Fatores de Coagulação Sanguínea/análise , Lipídeos/sangue , Infarto do Miocárdio/sangue , Apolipoproteínas/sangue , Feminino , Seguimentos , Cardiopatias/mortalidade , Humanos , Masculino , Análise Multivariada , Infarto do Miocárdio/epidemiologia , Prognóstico , Modelos de Riscos Proporcionais , Recidiva , Fatores de Risco , Caracteres SexuaisRESUMO
The obese Zucker diabetic fatty male rat (ZDF/Gmi¿trade mark omitted¿-fa) has become a widely used animal model of NIDDM, in contrast to the obese ZDF females that rarely develop NIDDM. However, preliminary observations suggest that obese ZDF females may become diabetic on high-fat diets. Therefore, we studied the effect of dietary fat on development of NIDDM, dyslipidemia, and alterations in organ-specific serum panels in obese ZDF males and females. Results indicated different effects of dietary fat-content on development of diabetes in males and females. Males, even on low fat-content diets, developed diabetes but the process was accelerated as a function of dietary fat-content, whereas only the highest fat-content diet induced development of NIDDM in obese ZDF females. Additionally, triglyceride/apolipoprotein B ratios demonstrated gender-specific differences in the nature of circulating lipoprotein particles independent of diabetic state with values for females approximately twice those of males indicating more highly triglyceride-enriched lipoprotein particles in females. We conclude that the obese ZDF female rat has the potential to become an important animal model of NIDDM especially in women where few models currently exist.
Assuntos
Diabetes Mellitus Tipo 2/etiologia , Gorduras na Dieta/farmacologia , Obesidade/complicações , Ratos Zucker/fisiologia , Caracteres Sexuais , Animais , Apolipoproteínas B/sangue , Modelos Animais de Doenças , Feminino , Hiperlipidemias/etiologia , Masculino , Obesidade/sangue , Ratos , Triglicerídeos/sangueRESUMO
The cDNA encoding rat betaine-homocysteine S-methyltransferase (BHMT) was isolated through production of monoclonal antibodies against protein fractions enriched with apolipoprotein B (apo B)-mRNA-editing complexes. BHMT mRNA was expressed predominantly in liver, and also in kidney, but not in small intestine. In stable McArdle RH-7777 (McA) cell lines expressing differing levels of BHMT, the editing efficiency of apo B mRNA was unchanged. Evaluation of apo B-mRNA expression revealed that steady-state levels were increased significantly and in parallel with BHMT protein expression. The highest levels of BHMT mRNA and BHMT enzyme activity expressed in stably transfected McA cells were comparable with those found in rat hepatocytes. In contrast to the changes in apo B-mRNA abundance, levels of other apolipoprotein-encoding mRNAs and several liver-specific and ubiquitously expressed mRNAs were unchanged by BHMT expression. In the cell line expressing the highest level of BHMT, apo B-containing lipoprotein secretion was increased, indicating utilization of increased endogenous message. Results suggest that apo B-mRNA abundance in McA cells is related to the expression of BHMT, an enzyme important in homocysteine metabolism.
Assuntos
Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Metiltransferases/genética , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Betaína-Homocisteína S-Metiltransferase , DNA Complementar , Intestinos/enzimologia , Fígado/enzimologia , Dados de Sequência Molecular , Edição de RNA , Ratos , Células Tumorais CultivadasRESUMO
BACKGROUND: Thrombosis is a pivotal event in the pathogenesis of coronary disease. We hypothesized that the presence of blood factors that reflect enhanced thrombogenic activity would be associated with an increased risk of recurrent coronary events during long-term follow-up of patients who have recovered from myocardial infarction. METHODS AND RESULTS: We prospectively enrolled 1045 patients 2 months after an index myocardial infarction. Baseline thrombogenic blood tests included 6 hemostatic variables (D-dimer, fibrinogen, factor VII, factor VIIa, von Willebrand factor, and plasminogen activator inhibitor-1), 7 lipid factors [cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, lipoprotein(a), apolipoprotein (apo)A-I, and apoB], and insulin. Patients were followed up for an average of 26 months, with the primary end point being coronary death or nonfatal myocardial infarction, whichever occurred first. The hemostatic, lipid, and insulin parameters were dichotomized into their top and the lower 3 risk quartiles and evaluated for entry into a Cox survivorship model. High levels of D-dimer (hazard ratio, 2.43; 95% CI, 1.49, 3.97) and apoB (hazard ratio, 1.82; 95% CI, 1.10, 3.00) and low levels of apoA-I (hazard ratio, 1.84; 95% CI, 1.10, 3.08) were independently associated with recurrent coronary events in the Cox model after adjustment for 6 relevant clinical covariates. CONCLUSIONS: Our findings indicate that a procoagulant state, as reflected in elevated levels of D-dimer, and disordered lipid transport, as indicated by low apoA-1 and high apoB levels, contribute independently to recurrent coronary events in postinfarction patients.
Assuntos
Hemostasia , Infarto do Miocárdio/sangue , Infarto do Miocárdio/etiologia , Trombose/sangue , Trombose/complicações , Adulto , Idoso , Fator VII/metabolismo , Fator VIIa/metabolismo , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Fibrinogênio/metabolismo , Humanos , Insulina/sangue , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/fisiopatologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Estudos Prospectivos , Recidiva , Fatores de Risco , Trombose/fisiopatologia , Fator de von Willebrand/metabolismoRESUMO
Lipoprotein and apolipoprotein parameters were studied in the male Zucker diabetic fatty (ZDF) rat at 10 and 20 weeks of age, corresponding to hyperinsulinemic and insulinopenic type 2 diabetes mellitus, respectively. At both ages, ZDF rats had elevated serum triglycerides, free fatty acids, and corticosterone, whereas 20-week ZDF rats had reduced thyroid hormones. At 10 weeks, the hyperlipidemia was confined to elevations in pre-beta triglyceride-rich (d < 1.006 g/mL) lipoproteins. By 20 weeks, all lipoprotein density fractions were increased compared with lean rats, with substantial increases in both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol. In ZDF rats, there was a progressive increase in apolipoprotein B (apo B) from 1.9 times control at 10 weeks to three times control at 20 weeks. The increase in apo B was accompanied by a shift of apo B, particularly B100, from very-low-density lipoprotein (VLDL) into denser lipoproteins corresponding to intermediate-density lipoproteins plus LDLs (1.006 < d < 1.063 g/mL). In Zucker and 10-week ZDF rats, in the presence of hyperinsulinemia, the increase in serum apo B was predominantly apo B48 present in VLDL. By 20 weeks, when ZDF rats are insulinopenic, the mass ratio of B48:B100 shifted from 2.7 to 0.7. The shift was associated with a decrease in hepatic-edited apo B mRNA. Apo E increased in lean rats between 10 and 20 weeks of age. Although apo E also increased in ZDF rats, the increase by 20 weeks was less than that of lean rats. The molar ratio of apo E to B in VLDL was decreased in ZDF rats. In lean rats, greater than 50% of apo E was present in HDL, in contrast to ZDF rats, where less than 20% of apo E was present in HDL. VLDL apo E shifted to denser fractions by 20 weeks of age, similar to apo B. The apo C level was more than double compared with the level in lean rats and was redistributed from the HDL fraction to lipoprotein fractions containing apo B. Both apo A-I and apo A-IV levels more than doubled between 10 and 20 weeks in ZDF rats. The ZDF rat model may be useful in comparative studies of lipoproteins during diabetic progression from hyperinsulinemia to insulinopenia.
Assuntos
Diabetes Mellitus Experimental/sangue , Hiperglicemia/complicações , Hiperinsulinismo/complicações , Insulina/sangue , Lipoproteínas/sangue , Animais , Peso Corporal , Diabetes Mellitus Experimental/complicações , Lipoproteínas/classificação , Masculino , Tamanho do Órgão , Período Pós-Prandial , Ratos , Ratos ZuckerRESUMO
Apolipoprotein B (apoB) mRNA editing involves a site-specific cytidine to uridine transition catalyzed by the cytidine deaminase, APOBEC-1, in the context of and regulated by a multi-protein-containing editosome. ApoB mRNA editing in vivo is subject to tissue specific, developmental and metabolic regulation. We demonstrate for the first time that the amount of edited apoB mRNA in rat primary hepatocytes is markedly increased subsequent to transient treatment with ethanol in vitro. The apparent change in editing efficiency was dose-dependent (from 0.1%-2.4% initial ethanol dose) and occurred with rapid onset. The proportion of edited apoB mRNA was also markedly enhanced in a rat hepatoma cell line, McArdle RH7777 cells and in a stable McArdle cell line over-expressing APOBEC-1 by transient treatment with 2.5 % ethanol. In contrast, the apoB mRNA editing in a human hepatoma cell line, HepG2 cells and a stable HepG2 cell line over-expressing APOBEC-1 did not respond to ethanol treatment. The data support the possibility that editing activity is ethanol-responsive but suggest that this change is cell type-specific.
Assuntos
Apolipoproteínas B/genética , Etanol/farmacologia , Edição de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Relação Dose-Resposta a Droga , Etanol/administração & dosagem , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Oligodesoxirribonucleotídeos/genética , Ratos , Células Tumorais CultivadasRESUMO
The prothrombinase complex (factor [F]Xa, FVa, calcium ions, and lipid membrane) converts prothrombin to thrombin (FIIa). To determine whether plasma lipoproteins could provide a physiologically relevant surface, we determined the rates of FIIa production by using purified human coagulation factors, and isolated fasting plasma lipoproteins from healthy donors. In the presence of 5 nmol/L FVa, 5 nmol/L FXa, and 1.4 micromol/L prothrombin, physiological levels of very low density lipoprotein (VLDL) (0.45 to 0.9 mmol/L triglyceride, or 100 to 200 micromol/L phospholipid) yielded rates of 2 to 8 nmol Flla x L(-1) x s(-1) in a donor-dependent manner. Low density lipoprotein (LDL) and high density lipoprotein (HDL) also supported prothrombinase but at much lower rates (< or =1.0 nmol FIIa x L(-1) x s[-1]). For comparison, VLDL at 2 mmol/L triglyceride yielded approximately 50% the activity of 2X10(8) thrombin-activated platelets per milliliter. Although the FIIa production rate was slower on VLDL than on synthetic phosphatidylcholine/phosphatidylsenne vesicles (approximately 50 nmol FIIa x L(-1) x s[-1]), the prothrombin Km values were similar, 0.8 and 0.5 micromol/L, respectively. Extracted VLDL lipids supported rates approaching those of phosphatidylcholine/phosphatidylserine vesicles, indicating the importance of the intact VLDL conformation. However, the presence of VLDL-associated, factor-specific inhibitors was ruled out by titration experiments, suggesting a key role for lipid organization. VLDL also supported FIIa generation in an assay system comprising 0.1 nmol/L FVIIa; 0.55 nmol/L tissue factor; physiological levels of FV, FVIII, FIX, and FX; and prothrombin (3 nmol/L FIIa x L(-1) x s[-1]). These results indicate that isolated human VLDL can support all the components of the extrinsic coagulation pathway, yielding physiologically relevant rates of thrombin generation in a donor-dependent manner. This support is dependent on the intact lipoprotein structure and does not appear to be regulated by specific VLDL-associated inhibitors. Further studies are needed to determine the extent of this activity in vivo.
Assuntos
Coagulação Sanguínea/fisiologia , Enzimas/fisiologia , Lipoproteínas/sangue , Tromboplastina/metabolismo , Doadores de Sangue , Plaquetas/enzimologia , Fator VIIa/fisiologia , Fator Va/antagonistas & inibidores , Inibidores do Fator Xa , Humanos , Cinética , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/metabolismo , Lipoproteínas VLDL/farmacologia , Tromboplastina/fisiologiaRESUMO
Apolipoprotein B (apo B) secretion is reduced by insulin in rat hepatocytes. To evaluate possible mechanisms by which insulin action leads to inhibition of apo B secretion, we evaluated the effect of suppression of the protein-tyrosine phosphatase LAR on apo B secretion by McA-RH7777 (McA) rat hepatoma cells. A reduction in cellular LAR levels was accomplished by stable transfection of McA cells with LAR antisense cDNA. Previous studies indicate that LAR-antisense transfectants demonstrate increased insulin receptor signaling. In current studies, reduced LAR expression results in a 60% to 70% reduction in apo B secretion compared with null vector control. The reduction in apo B secretion correlated with a significant decrease in cellular apo B mRNA levels. Results suggests there is a relationship of protein tyrosine phosphorylation with regulation of apo B mRNA abundance in McA cells.
Assuntos
Apolipoproteínas B/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , Animais , Apolipoproteínas B/genética , Linhagem Celular , DNA Antissenso/farmacologia , DNA Complementar , Insulina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores , Transdução de Sinais , Células Tumorais CultivadasRESUMO
The effect of oleic acid (OA), stearic acid (SA) and elaidic acid (EA) on cellular and secreted apolipoprotein (apo) B was examined in McArdle RH-7777 (McArdle) hepatoma cells and in primary rat hepatocytes. ApoB secretion by McArdle cells was significantly inhibited by 20% in 8 h incubations in medium containing EA and SA and by 50% in medium containing OA. In contrast, apo B secretion and cellular apo B of primary rat hepatocytes was relatively unaffected by incubations in medium containing fatty acids. Both B100 and B48 secretion in McArdle wild type and B48 in apo B mRNA editing enzyme catalytic polypeptide transfectants expressing B48 were inhibited to a similar extent indicating an effect of OA on both apo B species. The effect of OA occurred without changes in cellular apo B or in apo B mRNA abundance suggesting a post-transcriptional mechanism. Time course studies indicate that the suppressive effect of OA requires 4 h of incubation suggesting the depletion of a limiting factor important in apoB secretion. By increasing the proportion of palmitic acid to OA in the medium, apoB secretion by McArdle cells was progressively restored to control levels implicating an unique role for newly synthesized saturated fatty acid.
Assuntos
Apolipoproteínas B/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Fígado/metabolismo , Ácido Oleico/farmacologia , Ácidos Esteáricos/farmacologia , Animais , Ácidos Oleicos , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
Insulin inhibits apolipoprotein B (apoB) secretion by primary rat hepatocytes through activation of phosphoinositide 3-kinase (PI 3-K). Current studies demonstrate that the PI 3-K inhibitor wortmannin inhibits both basal and insulin-stimulated PI 3-K activities. Wortmannin and LY 294002, two structurally distinct PI 3-K inhibitors, prevent insulin-dependent inhibition of apoB secretion in a dose-dependent manner. To link PI 3-K activation to insulin action on apoB, we investigated whether insulin induced localization of activated PI 3-K to the endoplasmic reticulum (ER), where apoB biogenesis is initiated. Insulin action results in a significant redistribution of PI 3-K to a low density microsome (LDM) fraction containing apoB protein and apoB mRNA. Insulin stimulates a significant increase in PI 3-K activity associated with insulin receptor substrate-1 as well as an increase in insulin receptor substrate-1/PI 3-K mass in LDM. Subfractionation of LDM on sucrose density gradients shows that insulin significantly increases the amount of PI 3-K present in an ER fraction containing apoB. Insulin stimulates PI 3-K activity in smooth and rough microsomes isolated from rat hepatocytes, the latter of which contain rough ER as demonstrated by electron microscopy. Studies indicate that 1) PI 3-K activity is necessary for insulin-dependent inhibition of apoB secretion by rat hepatocytes; 2) insulin action leads to the activation and localization of PI 3-K in an ER fraction containing apoB; and 3) insulin stimulates PI 3-K activity in the rough ER.
Assuntos
Apolipoproteínas B/metabolismo , Retículo Endoplasmático/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Androstadienos/farmacologia , Animais , Centrifugação com Gradiente de Concentração , Inibidores Enzimáticos/farmacologia , Proteínas Substratos do Receptor de Insulina , Microscopia Eletrônica , Microssomos Hepáticos/enzimologia , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , WortmaninaRESUMO
Hepatic apolipoprotein (apo) B RNA editing was examined in the genetically obese hyperinsulinemic and hypertriglyceridemic Zucker rat. In obese Zucker rats, apo B RNA editing was increased 42% relative to that in lean controls. Correspondingly, the proportion of serum triglyceride-rich lipoprotein containing apo B48 increased 4.7-fold in the obese Zucker rat. Quantification of hepatic total apo B mRNA showed no difference between obese Zucker and lean control rats. In contrast, the hepatic mRNA encoding APOBEC-1, the catalytic subunit of the RNA editing activity, demonstrated an increased abundance of 1.8-fold in obese Zucker rats versus lean controls.
Assuntos
Apolipoproteínas B/genética , Fígado/metabolismo , Obesidade/metabolismo , Edição de RNA , Desaminase APOBEC-1 , Animais , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Modelos Animais de Doenças , Hiperinsulinismo/sangue , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Hipertrigliceridemia/sangue , Hipertrigliceridemia/genética , Hipertrigliceridemia/metabolismo , Resistência à Insulina/genética , Masculino , Obesidade/genética , RNA Mensageiro/genética , Ratos , Ratos ZuckerRESUMO
A protein complex involved in apolipoprotein B (apoB) RNA editing, referred to as AUX240 (auxiliary factor containing p240), has been identified through the production of monoclonal antibodies against in vitro assembled 27S editosomes. The 240-kDa protein antigen of AUX240 colocalized with editosome complexes on immunoblots of native gels. Immunoadsorbed extracts were impaired in their ability to assemble editosomes beyond early intermediates and in their ability to edit apoB RNA efficiently. Supplementation of adsorbed extract with AUX240 restored both editosome assembly and editing activities. Several proteins, in addition to p240, ranging in molecular mass from 150 to 45 kDa coimmunopurify as AUX240 under stringent wash conditions. The activity of the catalytic subunit of the editosome APOBEC-1 and mooring sequence RNA binding proteins of 66 and 44 kDa could not be demonstrated in AUX240. The data suggest that p240 and associated proteins constitute an auxiliary factor required for efficient apoB RNA editing. We propose that the role of AUX240 may be regulatory and involve mediation or stabilization of interactions between APOBEC-1 subunits and editing site recognition proteins leading the assembly of the rat liver C/U editosome.
Assuntos
Apolipoproteínas B/biossíntese , Proteínas de Transporte/metabolismo , Fígado/metabolismo , Edição de RNA , RNA Mensageiro/metabolismo , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/isolamento & purificação , Fracionamento Celular , Reagentes de Ligações Cruzadas , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Cinética , Neoplasias Hepáticas Experimentais , Metionina/metabolismo , Peso Molecular , RNA Neoplásico/metabolismo , Ratos , Ratos Sprague-Dawley , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
Insulin inhibition of the secretion of apolipoprotein B (apo B) was studied in primary cultures of rat hepatocytes by using brefeldin A (BFA), an inhibitor of protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and by using the phosphatidylinositol 3-kinase (PI 3-K) inhibitor wortmannin. Incubation of hepatocytes with BFA (10 micrograms/ml) for 1 h inhibited the subsequent secretion of apo B, albumin and transferrin for up to 3 h. BFA treatment resulted in the time-dependent accumulation in cells of [14C]leucine-labelled proteins and apo B. Under conditions where insulin decreased total apo B (cell plus secreted), BFA blocked the insulin-dependent effect. These results suggest that export of apo B from the ER is a prerequisite for the observed insulin effect. Treatment of hepatocytes with wortmannin for 20 min abolished insulin inhibition of apo B secretion, suggesting that the insulin effect on the apo B pathway involves activation of PI 3-K. Enzyme inhibitor studies indicate that chymostatin and (+)-(2S,3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)-butylcarba moyl]-2- oxiranecarboxylate (E-64-c) partially block insulin effects on apo B compared with leupeptin, which had no discernible effect. The cell-permeable derivative of E-64-c, EST, and N-Ac-Leu-Leu-norleucinal (ALLN) were most effective in blocking insulin effects on apo B. These results suggest that insulin action on apo B in primary rat hepatocytes involves (1) vesicular movement of apo B from the ER; (2) activation of PI 3-K and (3) a cellular protease that is either a cysteine- or calcium-activated neutral protease.
Assuntos
Androstadienos/farmacologia , Apolipoproteínas B/metabolismo , Ciclopentanos/farmacologia , Inibidores Enzimáticos/farmacologia , Insulina/farmacologia , Fígado/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Transporte Biológico , Brefeldina A , Células Cultivadas , Ativação Enzimática , Hidrólise , Fígado/enzimologia , Fígado/metabolismo , Fosfatidilinositol 3-Quinases , Ratos , Ratos Sprague-Dawley , WortmaninaAssuntos
Apolipoproteínas B/isolamento & purificação , Cromatografia em Agarose/métodos , Animais , Apolipoproteína B-100 , Apolipoproteína B-48 , Apolipoproteínas B/sangue , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/isolamento & purificação , Cromatografia em Agarose/instrumentação , Cromatografia em Gel , Corantes , Eletroforese em Gel de Poliacrilamida , Coloide de Ouro , Humanos , Lipoproteínas/isolamento & purificação , Testes de Precipitina , Ratos , Dodecilsulfato de SódioRESUMO
This review has considered a number of observations obtained from studies of insulin in perfused liver, hepatocytes, transformed liver cells and in vivo and each of the experimental systems offers advantages. The evaluation of insulin effects on component lipid synthesis suggests that overall, lipid synthesis is positively influenced by insulin. Short-term high levels of insulin through stimulation of intracellular degradation of freshly translated apo B and effects on synthesis limit the ability of hepatocytes to form and secrete TRL. The intracellular site of apo B degradation may involve membrane-bound apo B, cytoplasmic apo B and apo B which has entered the ER lumen. How insulin favors intracellular apo B degradation is not known. An area of recent investigation is in insulin-stimulated phosphorylation of intracellular substrates such as IRS-1 which activates insulin specific cellular signaling molecules [245]. Candidate molecules to study insulin action on apo B include IRS-1 and SH2-containing signaling molecules. Insulin dysregulation in carbohydrate metabolism occurs in non-insulin-dependent diabetes mellitus due to an imbalance between insulin sensitivity of tissue and pancreatic insulin secretion (reviewed in Refs. [307,308]). Insulin resistance in the liver results in the inability to suppress hepatic glucose production; in muscle, in impaired glucose uptake and oxidation and in adipose tissue, in the inability to suppress release of free FA. This lack of appropriate sensitivity towards insulin action leads to hyperglycemia which in turn stimulates compensatory insulin secretion by the pancreas leading to hyperinsulinemia. Ultimately, there may be failure of the pancreas to fully compensate, hyperglycemia worsens and diabetes develops. The etiology of insulin resistance is being intensively studied for the primary defect may be over secretion of insulin by the pancreas or tissue insulin resistance and both of these defects may be genetically predetermined. We suggest that, in addition to effects in carbohydrate metabolism, insulin resistance in liver results in the inability of first phase insulin to suppress hepatic TRL production which results in hypertriglyceridemia leading to high levels of plasma FA which accentuate insulin resistance in other target organs. As recently reviewed [17,254] the role of insulin as a stimulator of hepatic lipogenesis and TRL production has been long established. Several lines of evidence support that insulin is stimulatory to the production of hepatic TRL in vivo. First, population based studies support a positive relationship between plasma insulin and total TG and VLDL [253]. Second, there is a strong association between chronic hyperinsulinemia and VLDL overproduction [309].(ABSTRACT TRUNCATED AT 400 WORDS)