RESUMO
Lung tissues from calves infected experimentally with Mycoplasma bovis were examined by immunohistochemistry and electron microscopy. All inoculated calves had dark red areas of consolidation affecting both left and right lungs, which were characterized microscopically by subacute purulent bronchiolitis with hyperplasia of the surrounding lymphoid tissue. Immunohistochemically, M. bovis antigen was detected on the surface and inside the cytoplasm of bronchiolar epithelial cells in the pneumonic foci. The antigen was also found in the cytoplasm of phagocytes at the margin of bronchiolar exudates. Electron microscopically, numerous organisms were demonstrated in the immunohistochemically-positive sites. These findings suggest that M. bovis organisms adhere to the bronchiolar epithelium and at least some of them invade the epithelium.
Assuntos
Bronquíolos/patologia , Doenças dos Bovinos/patologia , Animais , Bovinos , Mycoplasma bovis , Pneumonia por Mycoplasma/veterináriaRESUMO
We have studied a patient with extreme insulin resistance, acanthosis nigricans, and decreased erythrocyte insulin binding. EBV-transformed lymphocytes from this patient exhibited markedly reduced binding of 125I-insulin. Radioiodination of cell surface receptors followed by immunoprecipitation with anti-receptor antibodies revealed the presence of increased amounts of a 210-kD protein but no detectable alpha or beta subunits. Continuous labeling with 2-[3H]mannose revealed the synthesis of a 190-kD precursor and a 210-kD protein. The 210-kD protein was phosphorylated in an insulin-dependent manner at high insulin concentrations. These results suggest that in this patient the biosynthesis of 190-kD receptor precursor, its terminal glycosylation, and intracellular transport to the cell surface proceed normally, while proteolytic maturation to alpha and beta subunits does not occur. We postulate that this defect either results from mutation(s) within the insulin-receptor gene, which render the precursor resistant to cleavage, or from a defect in the receptor processing enzyme.
Assuntos
Resistência à Insulina , Linfócitos/metabolismo , Precursores de Proteínas/metabolismo , Receptor de Insulina/metabolismo , Acantose Nigricans/complicações , Acantose Nigricans/genética , Adulto , Linhagem Celular Transformada , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Transtornos do Crescimento/complicações , Transtornos do Crescimento/genética , Hirsutismo/complicações , Hirsutismo/genética , Humanos , Processamento de Proteína Pós-Traducional , SíndromeRESUMO
Prostacyclin-stimulating factor (PSF) acts on vascular endothelial cells to stimulate the synthesis of the vasodilatory molecule prostacyclin (PGI2). We have examined the expression, regulation, and hemodynamic bioactivity of PSF both in whole retina and in cultured cells derived from this tissue. PSF was expressed in all retinal cell types examined in vitro, but immunohistochemical analysis revealed PSF mainly associated with retinal vessels. PSF expression was constitutive in retinal pericytes (RPCs) but could be modulated in bovine retinal capillary endothelial cells (RECs) by cell confluency, hypoxia, serum starvation, high glucose concentrations, or inversely by soluble factors present in early vs. late retinopathy, such as TGF-beta, VEGF, or bFGF. In addition, RPC-conditioned media dramatically increased REC PGI2 production, a response inhibited by blocking PSF with a specific antisense oligodeoxynucleotide (ODN). In vivo, PGI2 increased retinal blood flow (RBF) in control and diabetic animals. Furthermore, the early drop in RBF during the initial weeks after inducing diabetes in rats, as well as the later increase in RBF, both correlated with levels of retinal PSF. RBF also responded to treatment with RPC-conditioned media, and this effect could be partially blocked using the antisense PSF ODN. We conclude that PSF expressed by ocular cells can induce PGI2, retinal vascular dilation, and increased retinal blood flow, and that alterations in retinal PSF expression may explain the biphasic changes in RBF observed in diabetes.
Assuntos
Epoprostenol/biossíntese , Retina/metabolismo , Animais , Sequência de Bases , Bovinos , Células Cultivadas , Primers do DNA/genética , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Hemodinâmica , Camundongos , Neovascularização Patológica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Retina/citologia , Vasos Retinianos/citologia , Vasos Retinianos/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Enhanced platelet functions have been demonstrated in patients with non-insulin-dependent diabetes mellitus (NIDDM). This study evaluated abnormalities in platelet signal transduction in diabetic patients, including turnover of phosphoinositides, mobilization of intracellular Ca2+, and phosphorylation of 20,000- and 47,000-Mr proteins (P20 and P47). Washed platelets were obtained from 6 patients with NIDDM whose platelet aggregation rates were abnormally elevated (DM-A group), 11 NIDDM patients with normal platelet aggregation rates (DM-B group), and 8 age-matched healthy control subjects. The mass and specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), and phosphatidic acid (PA) in 32P-labeled platelets were not different among the three groups. Hydrolysis of PIP2, PIP, and PI; accumulation of PA; and phosphorylation of P20 in platelets stimulated by 0.05 U/ml thrombin were significantly increased in the DM-A group compared with the control or DM-B group. There was no difference in P47 phosphorylation among the three groups. On the contrary, P20 and P47 phosphorylation induced by 50 nM of 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, was significantly decreased in the DM-A group. Additionally, the intracellular free Ca2+ concentration [( Ca2+]i) was measured with the fluorescent Ca2+ indicator fura 2. Although the basal [Ca2+]i value was similar in the three groups, the rise in [Ca2+]i induced by 0.05 U/ml thrombin in the presence and the absence of extracellular Ca2+ was significantly higher in the DM-A group than the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Cálcio/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Fosfatidilinositóis/metabolismo , Fosfoproteínas , Adulto , Plaquetas/fisiologia , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Quinase de Cadeia Leve de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/fisiologia , Miosinas/metabolismo , Ácidos Fosfatídicos/metabolismo , Radioisótopos de Fósforo , Fosforilação , Proteína Quinase C/metabolismo , Proteína Quinase C/fisiologia , Transdução de Sinais/fisiologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
With isolated perfused pancreases from normal and diabetic model rats, we studied alterations of the secretion of islet amyloid polypeptide, or amylin, which has been recently identified as a major component of amyloid deposits in the pancreatic islets of patients with non-insulin-dependent diabetes mellitus. Neonatal (n) Wistar-King albino rats given streptozocin (STZ) on the 2nd (n2STZ) or 5th (n5STZ) neonatal day exhibited moderate and marked elevations, respectively, of plasma glucose and HbA1 as adults compared with control rats given the vehicle. The release of amylin from the perfused pancreases in response to glucose and arginine paralleled that of insulin in all three groups. However, the molar ratio of secreted amylin to insulin in response to 16.7 mM glucose by n5STZ pancreases (6.55 +/- 0.71%) was significantly greater than that for either n2STZ (1.71 +/- 0.24%, P less than 0.05) or the control (0.60 +/- 0.03%, P less than 0.05) pancreases. The secreted amylin-insulin ratio of n2STZ pancreases also was significantly greater than that of the controls (P less than 0.05). The increased amylin-insulin molar ratios of both n2STZ and n5STZ pancreases also occurred during infusions of 33.3 mM glucose and 10 mM arginine. These findings suggest that amylin secretion may be preserved in diabetic rats with reduced beta-cell mass and that hyperglycemia may increase amylin production independently of that of insulin, which may be significant in the pathogenesis of non-insulin-dependent diabetes mellitus.
Assuntos
Amiloide/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Estreptozocina/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Arginina/farmacologia , Glucose/farmacologia , Técnicas In Vitro , Secreção de Insulina , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Masculino , Perfusão , Radioimunoensaio , Ratos , Ratos EndogâmicosRESUMO
Amylin is a 37-amino acid peptide isolated from the islet amyloid of patients with non-insulin-dependent diabetes mellitus. The isolated perfused normal rat pancreas was used to evaluate the effects of glucose and insulin secretagogues, such as arginine, beta-hydroxybutyrate, and gliclazide, on amylin secretion. Glucose and the other stimulants tested elicited a significant release of amylin from the rat pancreas in a biphasic pattern, similar to that of insulin. Dose-response studies of the glucose-induced release of amylin and insulin revealed that they possessed a similar dependency on glucose. However, the release of amylin induced by high concentrations of glucose was partially dissociated from that of insulin; that is, the amylin-insulin molar ratios induced by 22.2 and 33.3 mM glucose (1.11 +/- 0.05 and 1.05 +/- 0.04%, respectively) were significantly higher than those induced by 16.7 mM glucose (0.90 +/- 0.04%, P less than 0.01 vs. 22.2 mM glucose, P less than 0.05 vs. 33.3 mM glucose). Additionally, when the basal concentration of glucose in the perfusate was increased from 5.6 to 11.1 mM, the response of amylin was unchanged. These data suggest that amylin may be an islet hormone whose abundant response to high concentrations of glucose might contribute to the oversecretion of amylin in the hyperglycemia that accompanies diabetes mellitus.
Assuntos
Amiloide/metabolismo , Arginina/farmacologia , Gliclazida/farmacologia , Glucose/farmacologia , Hidroxibutiratos/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Ácido 3-Hidroxibutírico , Animais , Técnicas In Vitro , Secreção de Insulina , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Masculino , Perfusão , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
We investigated the effects of glucose on specific D-alpha-tocopherol binding to cultured bovine aortic endothelial cells. Our results confirmed that cultured bovine aortic endothelial cells have specific binding sites for D-alpha-tocopherol. These binding sites exhibited time- and temperature-dependent saturation. The specific binding affinity of D-alpha-tocopherol was significantly lower in endothelial cells cultured in high concentrations of glucose (16.8 or 22.4 mM) for > 7 days compared with cells cultured in a physiological glucose concentration (5.6 mM). No significant reduction occurred in D-alpha-tocopherol binding when 11.1 mM mannitol was added to cells cultured in 5.6 mM glucose. The addition of an aldose reductase inhibitor (ICI-128436, Statil) did not significantly affect the high-glucose-induced reduction of D-alpha-tocopherol binding, although it reduced sorbitol levels in the cells compared with those from cells cultured in high concentrations of glucose. Moreover, significantly higher amounts of lipid peroxides were produced in aortic endothelial cells cultured in high concentrations of glucose (16.8 or 22.4 mM) for > 3 days compared with cells cultured in a physiological concentration of glucose. These results indicate that high concentrations of glucose reduce D-alpha-tocopherol binding through mechanisms independent of putative osmotic effects of sorbitol accumulation in the cells. Possible mechanisms include glycation of protein or oxidative damage of cells and/or redox and metabolic imbalances associated with increased flux of glucose via the sorbitol pathway. A glucose-mediated reduction in D-alpha-tocopherol binding could diminish the beneficial effects of D-alpha-tocopherol to vascular endothelial cells and thereby may increase the vascular toxicity of hyperglycemia in diabetes mellitus.
Assuntos
Glucose/farmacologia , Túnica Íntima/efeitos dos fármacos , Vitamina E/metabolismo , Aldeído Redutase/antagonistas & inibidores , Animais , Aorta Torácica , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Epoprostenol/biossíntese , Radicais Livres/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Concentração Osmolar , Ftalazinas/farmacologia , Temperatura , Fatores de Tempo , Túnica Íntima/citologia , Túnica Íntima/metabolismoRESUMO
Hyperglycemia and diabetes have been shown to increase diacylglycerol (DAG) level and activate protein kinase C (PKC) activity in the vascular tissues, possibly altering vascular function. We have characterized the effects of D-alpha-tocopherol (vitamin E) on PKC activities and DAG levels in rat aortic smooth muscle cells (ASMCs) cultured with elevated glucose levels as well as in the vascular tissues obtained from control and diabetic rats. In ASMCs, the specific PKC activity from the membraneous fraction and total DAG level were increased by 31 +/- 4% (P < 0.05) and 50 +/- 7% (P < 0.05), respectively, when the glucose levels were changed from 5.5 to 22 mmol/l. The addition of D-alpha-tocopherol and another lipophilic antioxidant, probucol, prevented the glucose-stimulated increases in DAG level and PKC activity. By immunoblotting studies, D-alpha-tocopherol treatment was able to reduce the enhancement of PKC beta II isoform in the membraneous fraction isolated from ASMCs. Comparing streptozotocin-induced diabetic rats with their nondiabetic controls, both membraneous-specific PKC activities and total cellular DAG levels were increased in aorta by 162% (P < 0.05) and 60% (P < 0.05), respectively. Intraperitoneal injection of D-alpha-tocopherol (40 mg/kg) every other day prevented the increases in membraneous-specific PKC activities and total DAG levels in parallel with a significant increase of D-alpha-tocopherol contents in the aorta and plasma. These findings have demonstrated that D-alpha-tocopherol can prevent the activation of PKC activities in the vascular cells and tissues induced by hyperglycemia by lowering DAG levels, possibly via its antioxidant effect.
Assuntos
Vasos Sanguíneos/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diglicerídeos/metabolismo , Proteína Quinase C/efeitos dos fármacos , Vitamina E/farmacologia , Animais , Aorta/efeitos dos fármacos , Vasos Sanguíneos/enzimologia , Vasos Sanguíneos/metabolismo , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Glucose/antagonistas & inibidores , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Prostacyclin (PGI2) synthesis by vascular endothelial cells (ECs) decreases in diabetic subjects, possibly leading to the development of diabetic angiopathy, such as that seen in atherosclerosis. We recently found a novel bioactive peptide, prostacyclin-stimulating factor (PSF), which stimulates PGI2 synthesis by cultured aortic ECs. Our previous studies demonstrated that PSF is dominantly expressed by arterial smooth muscle cells (SMCs). In the present study, we found PSF to exist in the SMCs of human coronary arteries by means of immunohistochemical methods. Human coronary arteries obtained from autopsies were divided into four subgroups, with or without NIDDM and/or myocardial infarction. Immunostaining for PSF was performed by the avidin-biotin peroxidase complex method using a purified anti-PSF antibody, and the immunostaining for PSF was assessed semiquantitatively. PSF staining was markedly reduced in coronary arterial SMCs from patients with NIDDM and/or myocardial infarction. In addition, the effect of a high glucose culture on PSF mRNA expression and PSF production in bovine aortic SMCs was examined by immunocytochemical staining and both Western and Northern blot analyses. The immunostaining and immunoblot band for PSF also significantly decreased when bovine aortic SMCs were cultured with high concentrations of glucose. Furthermore, as compared with the SMCs cultured with a physiological glucose concentration, the density ratio of PSF mRNA to 28S rRNA expression significantly decreased when the SMCs were cultured with high concentrations of glucose. These results strongly suggest that the decreased PSF production may thus results in a decreased production of PGI2 in the coronary artery, thus leading to the development of both diabetic macroangiopathy and atherosclerosis.
Assuntos
Arteriosclerose/metabolismo , Fatores Biológicos/farmacologia , Vasos Coronários/química , Diabetes Mellitus Tipo 2/metabolismo , Epoprostenol/biossíntese , Imuno-Histoquímica , Adulto , Idoso , Idoso de 80 Anos ou mais , Aorta , Western Blotting , Células Cultivadas , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/química , RNA Mensageiro/metabolismoRESUMO
Gap junction is thought to have a crucial role in maintaining tissue homeostasis. We examined the effect of a high glucose level on gap junctional intercellular communication (GJIC) activity in cultured vascular smooth muscle cells (VSMCs) using the fluorescent dye transfer method. After a 48-h incubation with 22 mmol/l glucose (high glucose level), GJIC activity of VSMCs was significantly reduced compared with incubation with 5.5 mmol/l glucose (normal glucose level) (P < 0.05). Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA; 5 x 10(-8) mol/l), a protein kinase C (PKC) activator, for 1 h also reduced GJIC activity (P < 0.01). In addition, treatment of the cells with calphostin C, a specific PKC inhibitor, for 3 h completely restored the GJIC activity inhibited by the high glucose level. Western blot analysis showed that connexin 43 (Cx43), which is the major functional protein of gap junction, is present in multiphosphorylated forms: a nonphosphorylated form (P0) and phosphorylated forms (P1, P2, and P3). Incubation of VSMCs with a high glucose level significantly increased the density ratio of P3/P0 compared with a normal glucose level (P < 0.05). Similarly, treatment of the cells with TPA significantly increased the P3/P0 ratio compared with controls (P < 0.01). In addition, the increase in the P3/P0 density ratio induced by a high glucose level was restored to the control level by both staurosporine and calphostin C. These results suggest that the high glucose level induced the inhibition of GJIC activity in cultured VSMCs through excessive phosphorylation of Cx43, mediated by PKC activation. This may contribute to the development of the macroangiopathy associated with diabetes.
Assuntos
Aorta Torácica/fisiologia , Comunicação Celular/fisiologia , Conexina 43/metabolismo , Junções Comunicantes/fisiologia , Glucose/farmacologia , Músculo Liso Vascular/fisiologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Bovinos , Comunicação Celular/efeitos dos fármacos , Células Cultivadas , Conexina 43/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática , Junções Comunicantes/efeitos dos fármacos , Manitol/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Naftalenos/farmacologia , Fosforilação , Proteína Quinase C/metabolismo , Estaurosporina/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
After induction of diabetes with streptozocin (STZ-D) in rats, we measured vasoactive intestinal polypeptide (VIP) content in sciatic nerve and spinal cord obtained from nondiabetic, untreated STZ-D, and insulin-treated STZ-D rats. Eight weeks after the onset of diabetes, caudal nerve conduction velocity (NCV) in the untreated STZ-D rats (n = 13) was slower than in the controls (n = 11; mean +/- SE 30.9 +/- 0.6 vs. 41.4 +/- 1.8 m/s, P less than 0.001). The decrease in NCV was less marked in the insulin-treated STZ-D rats (n = 11; 36.3 +/- 0.9 m/s, P less than 0.05 vs. control). VIP content in sciatic nerve decreased in the untreated STZ-D rats (1.33 +/- 0.23 ng/g wet wt) compared with the other groups (control, 3.10 +/- 0.44, P less than 0.01; insulin-treated STZ-D, 2.44 +/- 0.55, P less than 0.05). However, in spinal cord, VIP content was not significantly different among the three groups. The VIP levels in sciatic nerve showed a positive correlation with NCV (r = 0.430, P less than 0.01). In addition, an inverse correlation between VIP levels and blood glucose levels was observed (r = -0.5624, P less than 0.001). NCV was also inversely correlated with blood glucose levels (r = -0.7662, P less than 0.001). Together with a previous morphological study, these findings suggest a possible causal relationship between reduced VIP content and diabetic neuropathy.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Neuropatias Diabéticas/metabolismo , Nervo Isquiático/metabolismo , Medula Espinal/metabolismo , Peptídeo Intestinal Vasoativo/análise , Animais , Glicemia/análise , Peso Corporal , Insulina/uso terapêutico , Masculino , Condução Nervosa , Radioimunoensaio , Ratos , Ratos EndogâmicosRESUMO
We recently purified and cloned a new protein that stimulates the synthesis of prostacyclin (PGI2) by the vascular endothelial cells (ECs). We have termed this protein "PGI2-stimulating factor" (PSF). The present study evaluated the expression of PSF mRNA in tissues of Wistar rats, including the kidneys of rats with streptozotocin-induced diabetes, and in cultured cells. Furthermore, we evaluated the presence of PSF in human sera and the immunohistochemical localization of PSF in tissues of patients obtained at autopsy. The latter included a coronary atherosclerotic lesion of a patient who died of acute myocardial infarction. PSF was observed by Northern blot analysis to be expressed in all rat tissues examined (brain, lung, liver, kidney, skeletal muscle, and fat tissue) and was expressed in cultured vascular ECs, smooth muscle cells (SMCs), and fibroblast cells (FCs). A decreased expression of PSF was observed in the kidneys of diabetic rats versus those of normal rats. The presence of PSF in human serum was confirmed by Western blot analysis. In humans, PSF was mainly localized in vascular ECs and SMCs of arterial media and in SMCs of bronchi. Reduced staining for PSF was found in an atherosclerotic versus a normal coronary artery of humans. PSF may be involved in the production of PGI2 in the vessel wall and may participate in the maintenance of vascular homeostasis. PSF abnormalities may be involved in the development of such vascular lesions as atherosclerosis and diabetic angiopathy.
Assuntos
Fatores Biológicos/fisiologia , Diabetes Mellitus Experimental/metabolismo , Epoprostenol/fisiologia , Sequência de Aminoácidos , Animais , Arteriosclerose/metabolismo , Sequência de Bases , Vasos Coronários/metabolismo , Expressão Gênica , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , Ratos , Ratos Wistar , Distribuição TecidualRESUMO
Recent studies have revealed that vascular cells can produce reactive oxygen species (ROS) through NAD(P)H oxidase, which may be involved in vascular injury. However, the pathological role of vascular NAD(P)H oxidase in diabetes or in the insulin-resistant state remains unknown. In this study, we examined the effect of high glucose level and free fatty acid (FFA) (palmitate) on ROS production in cultured aortic smooth muscle cells (SMCs) and endothelial cells (ECs) using electron spin resonance spectroscopy. Exposure of cultured SMCs or ECs to a high glucose level (400 mg/dl) for 72 h significantly increased the free radical production compared with low glucose level exposure (100 mg/dl). Treatment of the cells for 3 h with phorbol myristic acid (PMA), a protein kinase C (PKC) activator, also increased free radical production. This increase was restored to the control value by diphenylene iodonium, a NAD(P)H oxidase inhibitor, suggesting ROS production through PKC-dependent activation of NAD(P)H oxidase. The increase in free radical production by high glucose level exposure was completely restored by both diphenylene iodonium and GF109203X, a PKC-specific inhibitor. Exposure to palmitate (200 micromol/l) also increased free radical production, which was concomitant with increases in diacylglycerol level and PKC activity. Again, this increase was restored to the control value by both diphenylene iodonium and GF109203X. The present results suggest that both high glucose level and palmitate may stimulate ROS production through PKC-dependent activation of NAD(P)H oxidase in both vascular SMCs and ECs. This finding may be involved in the excessive acceleration of atherosclerosis in patients with diabetes and insulin resistance syndrome.
Assuntos
Vasos Sanguíneos/metabolismo , Ácidos Graxos não Esterificados/farmacologia , Glucose/farmacologia , NADPH Oxidases/metabolismo , Proteína Quinase C/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Aorta , Vasos Sanguíneos/efeitos dos fármacos , Bovinos , Células Cultivadas , Espectroscopia de Ressonância de Spin Eletrônica , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ácidos Graxos não Esterificados/administração & dosagem , Glucose/administração & dosagem , Indóis/farmacologia , Maleimidas/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , NADPH Oxidases/antagonistas & inibidores , Oniocompostos/farmacologia , Ácido Palmítico/farmacologia , Proteína Quinase C/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
To examine the characteristic features of risk factors for macroangiopathy (MA) in nonobese Japanese NIDDM patients, 899 NIDDM patients with and without MA were registered from 40 facilities. Of these, 386 subjects were identified as having any form of MA (total MA); these included 211 with ischemic heart disease (IHD), 163 with cerebrovascular disease (CVD), and 77 with peripheral vascular disease (PVD). Univariate analyses revealed the following common risk factors for total MA, IHD, CVD, and PVD: age, hypertension, systolic blood pressure (sBP) or diastolic blood pressure (dBP), duration of diabetes, diabetic microangiopathy (retinopathy, nephropathy, and neuropathy), low HDL cholesterol level, and higher LDL cholesterol/HDL cholesterol ratio. Additional significant risk factors for specific conditions were also identified, respectively, as male sex for total MA, IHD, and PVD, smoking for IHD and PVD, and high fasting plasma glucose level for total MA and CVD. With stepwise multivariate logistic regression analysis, older age, duration of diabetes, smoking, and low LDL cholesterol/HDL cholesterol ratio were identified as significant and independent risk factors for total MA, IHD, CVD, and PVD. Other risk factors identified were high dBP for IHD, CVD, and PVD, high sBP for total MA, and low BMI for PVD. These results clearly demonstrated that duration of diabetes, smoking, hypertension, and dyslipidemia are major risk factors for MA in NIDDM patients. Since the mean BMI was similar for both groups (approximately 23 kg/m2) and there were no significant differences in immunoreactive insulin levels before and after 75-g oral glucose challenge testing, obesity and hyperinsulinism at the time of the analyses were not considered to play an important role for the pathogenesis of MA in Japanese NIDDM patients. By using the chi 2 test, cutoff points were determined for six of the most commonly measured risk factors. The cutoff point was the level beyond which a significantly higher prevalence of MA occurred. The cutoff points (rounded slightly upward in some cases) for fasting plasma glucose, sBP, dBP, serum total cholesterol level, serum triglyceride level, and BMI were 140 mg/dl, 140 mmHg, 80 mmHg, 180 mg/dl, 120 mg/dl, and 23 kg/m2, respectively. When these cutoff points were used as control criteria, the prevalence of MA was significantly lower in subjects whose risk factor measurements remained under the proposed control criteria for four or more of the six variables. In conclusion, in nonobese NIDDM patients, age, hypertension, and dyslipidemia were found to be risk factors for MA. Duration of diabetes was also demonstrated as an independent risk factor, indicating the close association of deranged glucose metabolism with the pathogenesis of MA in NIDDM patients. It seems to be crucial to control these risk factors for the prevention of MA in NIDDM patients.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/epidemiologia , Idoso , Pressão Sanguínea , Colesterol/sangue , Feminino , Fibrinogênio/metabolismo , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Análise de Regressão , Fatores de Risco , Triglicerídeos/sangueRESUMO
Mutation of the insulin receptor gene can compromise the ability of the receptor to mediate insulin action. A homozygous point mutation that results in the substitution of histidine for arginine 252 in the insulin receptor alpha-subunit has now been identified by polymerase chain reaction and single stranded conformational polymorphism analysis in a 20-yr-old Japanese woman with type A syndrome and severe insulin resistance. The proband's consanguineous parents (diabetic mother and normal father) and her sister (impaired glucose tolerance), each of whom showed an exaggerated insulin response to an oral glucose load, were heterozygous for this mutation. Her brother showed a normal insulin response and lacked the mutation, as did 50 healthy Japanese control subjects. The chronic sc administration of insulin-like growth factor I (IGF-I) improved the patient's hyperglycemia and corrected certain metabolic abnormalities over a 9-month period, even though the binding of 125I-labeled IGF-I to her cultured fibroblasts was decreased by 40% relative to that to cells from healthy controls. Studies of the binding of 125I-labeled insulin to the proband's cultured fibroblasts, to COS-I cells transfected with complementary DNA encoding the mutant insulin receptor, and to partially purified mutant receptors revealed that the Arg252-->His mutation decreased both cell surface expression and the affinity for insulin for the receptor. These observations suggest that the homozygous Arg252-->His mutation is responsible for the type A insulin resistance of the proband, whereas in the heterozygous state, the mutation results in mild insulin resistance indistinguishable from that observed in noninsulin-dependent diabetes mellitus.
Assuntos
Resistência à Insulina/genética , Fator de Crescimento Insulin-Like I/uso terapêutico , Receptor de Insulina/genética , Adolescente , Adulto , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Códon , DNA/genética , Feminino , Fibroblastos/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Dados de Sequência Molecular , Mutação , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita SimplesRESUMO
We previously described a case of familial hyperproinsulinemia, the fifth to be reported. In the present study we characterized the genetic defect carried by this family and demonstrated that it could be detected by polymerase chain reaction-single stranded conformational polymorphism. Since the serum proinsulin molecule from the propositus, a 63-yr-old Japanese man, was eluted on the same fraction of human proinsulin intermediate cleaved only at the B-C junction, we sequenced exon 3 of his insulin gene, including the C-A junction. A point mutation was discovered that changed codon 65 from arginine (CGT) to histidine (CAT) in one allele. This was the same point mutation as that described previously in three unrelated kindreds representing two races, consistent with the hypothesis that the dinucleotide sequence CpG may be a "hot spot" for mutations. Recently, developed polymerase chain reaction-single stranded conformational polymorphism proved useful in detecting this mutation in the family members. The daughter of the propositus and one of his two grandsons were also demonstrated to be heterozygous for this point mutation by this method.
Assuntos
DNA de Cadeia Simples/genética , Insulina/genética , Mutação Puntual , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético , Proinsulina/sangue , Sequência de Bases , Eletroforese , Éxons , Humanos , Masculino , Pessoa de Meia-Idade , Conformação Molecular , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , LinhagemRESUMO
We have investigated the effect of glucose on the release of endothelin-1-like immunoreactivity (ET-1-LI) from cultured bovine aortic endothelial cells. Elevation of glucose concentrations in cultured media from 5.5 to 11.1 or 22.2 mM significantly stimulated ET-1-LI release from cultured endothelial cells. An aldose reductase inhibitor did not affect the high glucose-induced ET-1-LI release. These findings suggest the possibility that hyperglycemia in diabetic patients enhances ET-1-LI release at the local site of vascular endothelium, which might be involved in the developments of vascular complications and atherosclerosis.
Assuntos
Aorta/metabolismo , Glucose/farmacologia , Peptídeos/metabolismo , Animais , Aorta/efeitos dos fármacos , Bovinos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Endotelinas , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Glucose/metabolismo , Manitol/farmacologia , Ftalazinas/farmacologiaRESUMO
Reduced prostacyclin (PGI2) generation by the vascular wall shows a close relationship with the development of atherosclerosis. The present study found plasma-derived serum (PDS) to contain an activity which stimulated PGI2 production by cultured bovine aortic endothelial cells. Diabetic and aged patients with atherosclerotic disease were examined for abnormalities in that stimulatory activity in PDS. PDS obtained from both diabetics (NIDDM) and aged patients showed a significant reduction in the stimulation of PGI2 production by cultured bovine aortic endothelial cells compared with age-matched controls and young healthy volunteers, respectively. It was suggested that the reduced PGI2 stimulatory activity in PDS may be one of the pathogenic mechanisms of vascular lesions such as atherosclerosis in diabetics and aged humans.
Assuntos
Envelhecimento/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Endotélio Vascular/metabolismo , Epoprostenol/metabolismo , Adulto , Idoso , Células Cultivadas , Humanos , Masculino , Pessoa de Meia-Idade , Estimulação QuímicaRESUMO
A reduction in production of prostacyclin (PGI2) by the cells in the vascular wall may play a role in the pathogenesis of atherosclerosis in diabetic patients. The present study was undertaken to evaluate the effect of glucose on PGI2 production by endothelial cells in vitro. It was shown that PGI2 production by cultured bovine aortic endothelial cells was significantly reduced in the presence of a high concentration of glucose (300 mg/dl) compared with physiological concentrations of glucose (100 mg/dl). In contrast, no reduction in PGI2 production was observed in cells cultured with equimolar mannitol, suggesting that glucose itself, rather than the effect of osmolality, inhibited PGI2 production by cultured endothelial cells. In addition, a high concentration of glucose also inhibited the proliferation of cultured endothelial cells.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Epoprostenol/biossíntese , Glucose/farmacologia , Animais , Bovinos , Contagem de Células , Células Cultivadas , Meios de Cultura , Depressão Química , Dinoprostona/biossíntese , Endotélio Vascular/metabolismoRESUMO
We evaluated the effect of d-alpha-tocopherol (vitamin E) on the production of prostacyclin (PGI2) by cultured bovine aortic endothelial cells. Vitamin E at physiological doses significantly enhanced the production of PGI2 by aortic endothelial cells when added to the culture simultaneously with histamine, the Ca2+ ionophore A23187 (A23187), plasma-derived serum (PDS), or arachidonic acid. This effect was found to occur in a time- and dose-dependent manner, and the maximal enhancement was produced by 9.28 microM of vitamin E for 1 h incubations. Significantly lower amounts of lipid peroxides were measured in endothelial cells stimulated by 10% PDS with 9.28 microM of vitamin E than in those stimulated without vitamin E for over 24 h, although the stimulation during the initial 1 to 12 h period did not have a significant effect on lipid peroxide formation in cultured aortic endothelial cells. We also demonstrated that bovine aortic endothelial cells have specific binding sites for [3H]vitamin E that exhibited time- and temperature-dependent saturability. At 4 degrees C, the nonspecific binding was 8-12% of the total binding, and the specific binding reached equilibrium by 2 h. Specific binding increased with the concentration of [3H]vitamin E and became saturated at concentrations between 1.5 microM and 2.0 microM per 2.0 x 10(5) cells. Raising the unlabeled vitamin E concentration from 97.7 nM to 1,000 microM reduced the specific binding of 2.0 microM [3H]vitamin E.(ABSTRACT TRUNCATED AT 250 WORDS)