RESUMO
Receptor-binding kinetics and degradation of tyrosine A-14 and A-19 125I-labelled insulin was studied using cultured human lymphocytes. Receptor-binding ability of A-14 insulin was 1.5-times as high as that of A-19 insulin. Dissociation from receptors on lymphocytes showed no difference between these two labelled insulins. In association studies percent bound of A-14 insulin was 1.5-times as high as that of A-19 insulin at any time after incubation. These results suggested that lower binding affinity of A-19 insulin was due to decreased association rate, but not due to increased dissociation rate. Degradation of A-14 insulin by incubation media of lymphocytes was also 1.5-times as high as that of A-19 insulin.
Assuntos
Insulina/análogos & derivados , Receptor de Insulina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Insulina/metabolismo , Cinética , Linfócitos/metabolismo , TemperaturaRESUMO
Insulin analogues with different amino acids, including threonine, alanine, L-leucine, D-leucine, L-leucine amide, phenylalanine, tri-alanine, or desalanine, at the B-30 position were semisynthesized from pork insulin by the new enzymatic method. The order of ability of the insulin analogues to bind to anti-insulin sera was [AlaB-30] greater than desalanine greater than [ThrB-30] greater than [Ala-Ala-AlaB-30] greater than [D-LeuB-30], [Leu-NH2B-30],[PheB-30] greater than desoctapeptide greater than or equal to [LeuB-30]. The ability of insulin analogues with different amino acids at B-30 to bind to receptors, as well as their biologic potency tested with glucose uptake in isolated rat adipocytes, was comparable among the analogues. These results suggest that [LeuB-30]-insulin demonstrated the least immunoreactivity and has full activity in receptor binding and biologic effect, and that it may be useful for treatment of anti-insulin antibody-mediated insulin resistance.
Assuntos
Anticorpos Anti-Insulina , Resistência à Insulina , Insulina/análogos & derivados , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Complexo Antígeno-Anticorpo , Transporte Biológico Ativo/efeitos dos fármacos , Glicemia/metabolismo , Membrana Celular/metabolismo , Desoxiglucose/metabolismo , Feminino , Humanos , Insulina/metabolismo , Insulina/farmacologia , Cinética , Placenta/metabolismo , Gravidez , Ratos , Receptor de Insulina/metabolismo , Relação Estrutura-AtividadeRESUMO
Membrane preparations from monkey and pig hypothalami bound [125I]insulin specifically. The binding appeared to be greater by preparations from anterior than posterior portions of the pig hypothalamus. Binding was time dependent, and its dissociation was first order with a half-time at 22 degrees C of 14 min. Desalanine insulin was as effective as native insulin in inhibiting the binding of [125I]insulin, while proinsulin was less effective and desoctapeptide insulin still less effective in accord with their biologic activities. Binding by membranes from cortex and thalamus appeared to be less than from hypothalamus. [125I]insulin was infused into an arterial split monkey brain preparation to determine if insulin that was blood borne bound specifically to the primate hypothalamus. Half the brain was perfused with [125I]insulin alone and the other half with [125I]insulin plus an excess of unlabeled insulin. Radioautography showed specific binding of insulin localized to the median eminence, infundibular nucleus, and microvessels. Thus, the monkey and pig hypothalami bind insulin with characteristics similar to those reported for known target tissues for insulin. Furthermore, insulin from the blood stream binds to specific anatomical structures in the hypothalamus of the monkey.
Assuntos
Hipotálamo Anterior/metabolismo , Hipotálamo Posterior/metabolismo , Hipotálamo/metabolismo , Insulina/metabolismo , Animais , Membrana Celular/metabolismo , Córtex Cerebral/metabolismo , Haplorrinos , Insulina/análogos & derivados , Ligação Proteica , Receptor de Insulina/metabolismo , Suínos , Tálamo/metabolismoRESUMO
Glucose, insulin and somatostatin infusion over 2 hours effectively suppressed endogenous secretion of insulin, glucagon and growth hormones. Steady state plasma glucose level (SSPG) which should be inversely proportional to insulin sensitivity was obtained. In 6 adult-onset non-obese untreated diabetics, mean value of insulin sensitivity indices was significantly reduced compared with normal. In 5 insulin-treated diabetics and in 5 subjects with borderline glucose tolerance including 2 obese subjects, insulin sensitivity for glucose utilization was also significantly diminished.
Assuntos
Glicemia/análise , Diabetes Mellitus/sangue , Glucose/administração & dosagem , Insulina/administração & dosagem , Somatostatina/administração & dosagem , Diabetes Mellitus/tratamento farmacológico , Teste de Tolerância a Glucose , Humanos , Insulina de Ação Prolongada/uso terapêuticoRESUMO
Insulin sensitivity has been determined in primary nonobese diabetics and subjects with borderline glucose intolerance by a newly devised technique using glucose, insulin, and somatostatin infusion. Insulin sensitivity for glucose utilization was decreased in both adult- and juvenile-onset diabetics. Eight out of 88 diabetics had normal insulin sensitivty and were free from microvascular complications. In lean subjects with borderline glucose intolerance, insulin sensitivity was decreased, although an overlap with normal was noted. All obese subjects with borderline glucose intolerance had reduced insulin sensitivity. An inverse relationship was observed between insulin sensitivity and fasting plasma glucose (FPG), and a significant correlation was observed between FPG and steady state plasma glucose levels (SSPG; r = 0.57; P less than 0.001). Improvement of diabetic control in eight diabetics with sulfonylureas decreased SSPG in all (P less than 0.05), although normalization of SSPG was observed in only one. These results indicate that decreased sensitivity of insulin for peripheral glucose utilization may play an important role in the pathogenesis of diabetes. Elevated FPG levels reflect the presence of decreased insulin sensitivity in diabetes mellitus. Although decreased sensitivity is difficult to normalize, it can be enhanced by improving the diabetic control. An effort to maintain or enhance tissue insulin sensitivity in diabetes mellitus may be more important than attempts to stimulate the deteriorating pancreatic beta-cells to secrete more insulin.
Assuntos
Diabetes Mellitus/fisiopatologia , Glucose , Resistência à Insulina , Insulina , Somatostatina , Adolescente , Adulto , Idoso , Glicemia/análise , Diabetes Mellitus/tratamento farmacológico , Dieta para Diabéticos , Jejum , Glibureto/uso terapêutico , Humanos , Insulina/uso terapêutico , Pessoa de Meia-IdadeRESUMO
A new potentiator of insulin action, 5-[4-(1-methylcyclohexylmethoxy)-benzyl]thiazolidine-2,4-dione, was tested for activation of insulin action in vitro. The agent (50 mg X kg-1 X day-1) was orally administered to rats for 14 days and adipocytes from treated rats were used to assess insulin-binding, glucose uptake and glucose oxidation. In obese rats, the agent increased glucose uptake and oxidation without any change in insulin binding, whereas in lean or streptozotocin-treated rats it failed to increase glucose metabolism. Fat tissues were cultured with the agent for 24 h and were tested for insulin action. In the presence of insulin (10 ng/ml) in the culture media, the agent increased glucose oxidation in these cells without any change in insulin binding. However, without insulin in the culture media the agent did not increase glucose oxidation. Thus, the agent appeared to potentiate insulin action at the post receptor process.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Insulina/metabolismo , Obesidade/metabolismo , Receptor de Insulina/metabolismo , Tiazóis/farmacologia , Tiazolidinedionas , Tecido Adiposo/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Desoxiglucose/metabolismo , Sinergismo Farmacológico , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Insulina/sangue , Cinética , Ratos , Ratos EndogâmicosRESUMO
Two insulin genes of the NON mouse, an animal model of human non-obese, non-insulin-dependent diabetes mellitus, were isolated and characterized to examine the hypothesis that these genes are structurally different from those of normal mice. The NON mouse was found to have two non-allelic insulin genes, as does the normal mouse, and no structural differences were found between the normal and NON mouse in the nucleotide sequence of the insulin gene, including that of the 5'-transcriptional regulatory region, and in the deduced amino acid sequence. There was, however, an additional 113 bp sequence and seven point mutations in a further 5'-flanking region, and three point mutations in the 3'-flanking region of the insulin II gene. We conclude that reduced expression of insulin genes in the NON mouse is not due to the structural change in the known transcriptional regulatory region, although the effect on insulin II gene expression of an additional sequence upstream of the 5'-flanking region, as the negative regulatory factor, remains to be elucidated.
Assuntos
Diabetes Mellitus Tipo 2/genética , Proinsulina/genética , Precursores de Proteínas/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Deleção Cromossômica , Clonagem Molecular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Biblioteca Gênica , Insulina , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Dados de Sequência Molecular , Mapeamento por Restrição , Transcrição GênicaRESUMO
Bactericidal activity in compromised host's serum i.e. patients with cancer, the elderly, diabetes, was stronger than that in normal serum against Proteus mirabilis (P. mirabilis) but was weaker against Klebsiella pneumoniae (K. pneumoniae). Against Escherichia coli (E. coli), bactericidal activity on serum of patients with cancer was weaker in the following order, that in elderly serum, that in diabetic serum. Against Proteus vulgaris (P. vulgaris), bactericidal activity in elderly serum was similar to that in normal serum but was stronger than that in serum of patients with cancer. Against Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus), bactericidal activity in elderly serum and diabetic serum was similar to that in normal serum but was weaker than that in serum of patients with cancer. Piperacillin showed bactericidal activity in nutrient broth, normal serum and compromised host's serum at a concentration of 1/4 MIC against E. coli, P. mirabilis and P. aeruginosa. Aspoxicillin showed bactericidal activity in nutrient broth, and bactericidal or bacteriostatic activity in normal serum and serum of patients with cancer against E. coli and P. aeruginosa. While cefazolin and cefmetazole slightly inhibited the growth of bacteria in nutrient broth, they showed hardly any bactericidal activity in normal serum and compromised host's serum.
Assuntos
Antibacterianos/sangue , Atividade Bactericida do Sangue , Síndromes de Imunodeficiência/sangue , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Feminino , Humanos , Neoplasias Pulmonares/sangue , Masculino , Pessoa de Meia-Idade , Teste Bactericida do Soro , beta-LactamasRESUMO
A modified erythrocyte insulin receptor assay was developed. The major advantage of our method is the smaller amount of blood necessary for the assay and consequent simple handling in the binding assay. Three ml of blood is enough for this assay, making it possible to assess insulin receptors in a group of pediatric patients. There was a close relationship between mononuclear cells and erythrocyte insulin binding assays (r = 0.900, p < 0.05). With this method insulin binding studies were done on normal subjects and adult onset non-obese diabetics. Decreased insulin binding was seen in the diabetic group, and this was due to a decreased number of insulin receptors. This erythrocyte insulin receptor assay can be useful tool for the study of the mechanism of insulin resistance.
Assuntos
Diabetes Mellitus/sangue , Eritrócitos/metabolismo , Insulina/metabolismo , Receptor de Insulina/análise , Animais , Preservação de Sangue , Humanos , Monócitos/metabolismo , SuínosRESUMO
AIM: Diabetic xerostomia is a typical syndrome in diabetic complication. We have reported that salivatin (salivary peptide P-C) derived from human saliva potentiates glucose-stimulated insulin release and inhibits arginine-stimulated glucagon release. The present study is aimed to gain further evidence on the physiological role by investigating the diabetic state-induced change in the amount of salivatin. METHODS: The amount of salivatin was measured in saliva taken from patients with type 2 diabetes with ELISA and with rabbit antiserum against human salivatin immunocytochemically in sections of parotid glands from streptozotocin-diabetic BALB/c mice. RESULTS: The amount of salivatin after a meal was reduced by diabetes in both human saliva and in the serous secretory granule of mouse parotid gland acinar cells. CONCLUSIONS: The above results suggest that salivatin lowers hyperglycaemia after meal and sustains the normal blood glucose levels by incretin-like mechanisms. The function may be damaged by diabetes, and this in turn might make the diabetes worse.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Peptídeos/metabolismo , Saliva/química , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Ingestão de Alimentos , Ensaio de Imunoadsorção Enzimática , Glucagon , Peptídeo 1 Semelhante ao Glucagon , Peptídeos Semelhantes ao Glucagon , Humanos , Hipoglicemiantes/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Imunoeletrônica , Glândula Parótida/patologia , Glândula Parótida/ultraestrutura , Fragmentos de Peptídeos/metabolismo , Peptídeos/análise , Período Pós-Prandial , Domínios Proteicos Ricos em Prolina , Proteínas Salivares Ricas em ProlinaRESUMO
We compared A-14 and A-19 125I-labelled insulin in receptor-binding and degradation. Percent receptor-binding of A-14 and A-19 125I-labelled insulin to 2.4 X 10(9)/ml erythrocytes after 210 min incubation at 15 degrees C was 7.8 and 4.9%, respectively. Percent insulin-receptor binding of A-14 insulin was 1.6 times greater than that of A-19 insulin. A similar result was obtained in an adipocytes insulin binding study. Percent receptor-binding of A-14 and A-19 insulin to 2 X 10(5)/ml fat cells after 30 min incubation in the above buffer was 3.9 and 2.4%, respectively. Degradation of A-14 and A-19 insulin in rat adipocytes was also studied by molecular sieve column chromatography. Isolated rat adipocytes were allowed to associate with A-14 and A-19 125I-insulin for 60 min at 37 degrees C, pH 8.0 in a HEPES-phosphate buffer, and then cells were separated from the buffer by centrifugation. After solubilization with triton X-100, both the solubilized cells and the incubation medium were applied to the Bio-Gel P-30 column to assess the insulin degradation. Degradation of A-14 125I-insulin by the isolated rat adipocytes was 1.6 times greater than that of A-19 125I-insulin. Furthermore, the peak which was thought to be intermediate degradation products of insulin was obtained between the peak of intact insulin and that of 125I-tyrosine. Such a peak of intermediates was much smaller in the incubation media than in the cell-associated materials.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Tecido Adiposo/metabolismo , Insulina/metabolismo , Receptor de Insulina/metabolismo , Animais , Técnicas In Vitro , Radioisótopos do Iodo , Masculino , Ratos , Ratos EndogâmicosRESUMO
We studied insulin degradation in human cultured lymphocytes (RPMI-1788 line) with a small but significant number of lysosomes under the electron microscope. Insulin degradation determined by the TCA solubility method was 64.6 +/- 1.2% (mean +/- SEM) at a trace concentration after the incubation with 2.0 x 10(7) cells (4.0 x 10(7) cells/ml) for 60 min at 37 degrees C. Because insulin degradation was 54.6 +/- 7.0% in the cell-free buffer in which 2.0 x 10(7) cells were previously incubated, most of the insulin was degraded outside of the cells. Gel filtration of the radioactive materials also revealed that most of the labeled insulin in the medium was degraded, and the main peak of the cell-associated radioactivities was intact labeled insulin. Chloroquine, a lysosomotropic agent, failed not only to increase insulin binding but also to decrease the insulin degradation. Other lysosomal protease inhibitors, antipain and leupeptin had also no effect on insulin degradation. In contrast, bacitracin (500 micrograms/ml) significantly decreased the insulin degradation analyzed by TCA solubility, receptor-rebinding, and the gel filtration method. These results suggest that insulin molecules are degraded by the enzymes leaked from the cells. The non-receptor mediated process, which is the bacitracin sensitive pathway, might be a general mechanism of insulin degradation in human cultured lymphocytes in vitro.
Assuntos
Insulina/metabolismo , Linfócitos/metabolismo , Receptor de Insulina/fisiologia , Bacitracina/farmacologia , Linhagem Celular , Cloroquina/farmacologia , Humanos , Linfócitos/ultraestrutura , Inibidores de Proteases/farmacologiaRESUMO
Yields of (14)CO(2) from [2-(14)C]mevalonate and [5-(14)C]mevalonate have been used by others to estimate the activity of the non-sterol-forming pathway, also called the mevalonate shunt pathway, and yields of (14)C in sterols have been used to estimate the activity of the sterol-forming pathway. Both these pathways operate following the conversion of carbon 1 of mevalonate to CO(2). The estimations of the shunt pathway contribution are dependent upon the fractions of carbons 2 and 5 of mevalonate that are oxidized to CO(2) in the Krebs cycle after leaving the pathway. Unless all of carbons 2 and 5 are oxidized to CO(2), the estimates are minimal. The metabolism of mevalonate has now been examined in slices of livers and kidneys from fasted rats and rats in diabetic ketosis. Yields of (14)CO(2) from [1-(14)C]mevalonate are used as the measure of the contributions of all the pathways by which carbon 1 of mevalonate is converted to CO(2). Yields of (3)H-labeled nonsaponifiable lipids from [5-(3)H]mevalonate are used as the measure of the sterol-forming pathway. The differences in these yields are then taken as the measure of the non-sterol-forming pathway or pathways. Yields of (14)CO(2) from [1-(14)C]mevalonate markedly exceeded the sum of the yields of (14)C in CO(2) and nonsaponifiable lipids from either [2-(14)C]mevalonate or [5-(14)C]mevalonate. Therefore, in liver and kidney, under the conditions of this study, either one or more pathways other than the shunt pathway, by which mevalonate can be metabolized to other than sterols, is operative to a marked degree, or estimates of the shunt pathway's contributions as judged by yields of (14)CO(2) from [2-(14)C]mevalonate and [5-(14)C]mevalonate are significantly underestimated.-Brady, P. S., W. C. Schumann, S. Ohgaku, R. F. Scofield, and B. R. Landau. Evidence for an underestimation of the shunt pathway of mevalonate metabolism in slices of livers and kidneys from fasted rats and rats in diabetic ketosis.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Cetoacidose Diabética/metabolismo , Rim/metabolismo , Fígado/metabolismo , Ácido Mevalônico/metabolismo , Animais , Dióxido de Carbono/análise , Radioisótopos de Carbono , Jejum , Feminino , Técnicas In Vitro , RatosRESUMO
Human [LeuB-24]- and [LeuB-25]-insulins were semi-synthesized from porcine insulin by an enzyme-assisted coupling method. The receptor-binding ability of [LeuB-24]- and [LeuB-25]-insulins was 30--48% and 2--5% respectively of that of human insulin. There was no significant difference in degradation between human insulin and these analogues on incubation with isolated adipocytes. The decreased affinity of these analogues was due to an increased dissociation rate rather than a change in the association rate of their binding to human cultured lymphocytes. The negative co-operative effect of [LeuB-24]- and [LeuB-25]-insulin was decreased to 50 and 1% respectively of that of human insulin at a concentration of 100 ng/ml. The ability of [LeuB-24]- and [LeuB-25]-insulin to stimulate 2-deoxyglucose uptake in isolated rat adipocytes was 35 and 4% respectively of that of human insulin. These analogues did not have an antagonistic effect on the biological activity of human insulin. The immunoreactivity of [LeuB-25]insulin was similar to that of porcine or human insulin, whereas [LeuB-24]insulin demonstrated decreased binding to anti-(porcine insulin) antibodies. These findings suggest that B-chain phenylalanine-25 residue is more crucial for receptor binding and negative co-operativity, whereas the B-chain phenylalanine-24 residue may play a more important role in binding to anti-insulin antibody.
Assuntos
Insulina/análogos & derivados , Receptor de Insulina/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Células Cultivadas , Desoxiglucose/metabolismo , Glucose/metabolismo , Humanos , Insulina/imunologia , Insulina/metabolismo , Insulina/farmacologia , Linfócitos/metabolismo , Especificidade da Espécie , SuínosRESUMO
We studied the effect of acute short-term exercise on insulin binding to erythrocytes in plasma with non-insulin dependent diabetes mellitus and normal controls. In the normal controls, insulin binding was increased immediately after the exercise and returned to the basal level at 30 min after the exercise. This increase was due to an affinity change without any change in the insulin receptor number. In contrast, insulin binding tended to decrease in the diabetics after the exercise, and this change was due to a decreased affinity without any change in the number of insulin receptors. These results indicate that the control mechanism of insulin receptor affinity in response to exercise is different in non-insulin dependent diabetics and normals.
Assuntos
Diabetes Mellitus/fisiopatologia , Eritrócitos/metabolismo , Insulina/sangue , Esforço Físico , Receptor de Insulina/metabolismo , Adulto , Feminino , Humanos , Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Radioimunoensaio , Ensaio RadioliganteRESUMO
We have investigated changes in insulin binding to erythrocytes in response to the oral administration of 500 mg of L-Dopa in ten healthy subjects. L-Dopa administration increases insulin binding from 5.18 +/- 0.14% (mean +/- SEM) to 6.18 +/- 0.34% (P less than 0.05) with concomitant increase in basal plasma growth hormone from 1.3 +/- 0.1 ng/ml to 14.4 +/- 4.8 ng/ml (P less than 0.05). The increase in insulin binding is due to increase in affinity of insulin receptor without changes in the number of insulin receptors. The plasma insulin and glucose did not show significant changes after L-Dopa administration. Direct incubation of L-Dopa with erythrocytes did not affect insulin binding to the cells. These results suggest that growth hormone may directly or indirectly induce acute alteration in affinity of insulin receptors on erythrocytes.
Assuntos
Hormônio do Crescimento/fisiologia , Insulina/metabolismo , Levodopa/farmacologia , Receptor de Insulina/efeitos dos fármacos , Adulto , Humanos , Masculino , Receptor de Insulina/metabolismoRESUMO
Insulin binding to erythrocytes from normal subjects over a wide range of ages (group I, cord blood; group II, Age 6M to 6 yrs; group III, 7 to 19 yrs; group IV, 20 to 59 yrs, group V, over 60 yrs of age) was determined using modified Gambhir's method and the effect of age and sex on the binding was evaluated. The highest insulin binding to cord blood erythrocytes was accounted for by the increased number of reticulocytes since there was a highly significant correlation between insulin binding and the reticulocyte count (r = 0.928, p less than 0.001). Group IV showed significantly higher binding than younger are groups II and III. The mean binding in group V was the highest among all age groups except for group I, although the differences failed to reach statistical significance. The receptor concentrations (R0) were comparable among groups except group I which had a slightly increased R0. The affinity of insulin receptors was the lowest in group I and II, and gradually increased with the age and a positive correlation (p less than 0.01) was obtained between age and Ke. This increase in affinity with age is considered an adaptive response to the intracellular metabolic derangement often seen in the aged. No effect of sex on insulin binding was seen before puberty. However, in normal adults insulin binding of the female tended to be lower than that of the male with slightly increased R0 and decreased affinity in the female. Thus, the slightly decreased binding of the female group was mainly accounted for by the decreased affinity although the difference in the affinity was not significant.