Insulin Receptor Assay Research Articles

Development of real-time reverse transcription polymerase chain reaction assays to quantify insulin-like growth factor receptor and insulin receptor expression in equine tissue

The insulin-like growth factor system (insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor and six insulin-like growth factor-binding proteins) and insulin are essential to muscle metabolism and most aspects of male and female reproduction. Insulin-like growth factor and insulin play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. In order to better understand the local factors that regulate equine physiology, such as muscle metabolism and reproduction (e.g., germ cell development and fertilisation), real-time reverse transcription polymerase chain reaction assays for quantification of equine insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were developed. The assays were sensitive: 192 copies/μL and 891 copies/μL for insulin-like growth factor 1 receptor, messenger ribonucleic acid and insulin receptor respectively (95% limit of detection), and efficient: 1.01 for the insulin-like growth factor 1 receptor assay and 0.95 for the insulin receptor assay. The assays had a broad linear range of detection (seven logs for insulin-like growth factor 1 receptor and six logs for insulin receptor). This allowed for analysis of very small amounts of messenger ribonucleic acid. Low concentrations of both insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were detected in endometrium, lung and spleen samples, whilst high concentrations were detected in heart, muscle and kidney samples, this was most likely due to the high level of glucose metabolism and glucose utilisation by these tissues. The assays developed for insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid expression have been shown to work on equine tissue and will contribute to the understanding of insulin and insulin-like growth factor 1 receptor physiology in the horse.

New, simple insulin-receptor assay with universal application to solubilized insulin receptors and receptors in broken and intact cells

New, simple insulin-receptor assay with universal application to solubilized insulin receptors and receptors in broken and intact cells

New, simple insulin-receptor assay with universal application to solubilized insulin receptors and receptors in broken and intact cells.

A new, simple insulin-receptor-binding assay has been devised. The assay is based on the separation of free and receptor-bound 125I-labeled insulin in 80% ethanol. It was found that the insulin-receptor complex was fully stable at this ethanol concentration, regardless of the source of the receptor employed. The assay has been evaluated with solubilized insulin receptors and membrane-bound receptors from human placenta and porcine liver as well as intact cells with the IM-9 cell line. The assay is simple, rapid, and has large capacity. Comparisons of the ethanol-based assay to the conventionally employed assays with polyethylene glycol or microfuge centrifugation for the separation of free and bound 125I-insulin revealed large discrepancies between the assays. The ethanol-based assay always appeared to provide a better separation. Microfuge centrifugation of placental membranes precipitated approximately 3% of the ethanol-precipitable insulin-receptor complex, while polyethylene glycol precipitation of solubilized insulin receptors varied between 40 and 80% of the ethanol precipitability, depending on the receptor concentration employed.

Down-Regulation of Insulin Receptors in Postnecrotic Cirrhosis of Liver*

Eighteen patients with postnecrotic cirrhosis of liver, including three patients who had had a portosystemic shunt operation, and 19 normal controls were studied. The tests performed included monocyte insulin receptor assay, iv glucose tolerance test, glucagon test, and insulin tolerance test. Insulin resistance was documented by the presence of fasting hyperglycemia and glucose intolerance together with hyperinsulinemia as well as resistance to exogenous insulin. The binding of [125I]insulin to monocyte insulin receptors was significantly decreased in cirrhotic patients compared with that in controls (P less than 0.02), and this was due to a significant decrease in the high affinity association constant (P less than 0.005). There was a significant negative correlation between the fasting insulin level and maximum [125I]insulin binding in cirrhotic patients (r = -0.8; P less than 0.02). Cirrhotics that had had a shunt operation showed a higher fasting insulin level, a greater insulin resistance, and a smaller maximum [125I]insulin binding to insulin receptors than those without shunt. All of these findings suggested a down-regulatory effect of hyperinsulinemia on the monocyte insulin receptors. An impaired glycemic response to glucagon was also found in cirrhotics, the exact mechanism of which remains to be elucidated. However, as the increases in plasma cAMP after glucagon were similar in cirrhotics and controls, the fault apparently did not lie in the glucagon receptor.

In vivo insulin sensitivity and insulin binding to erythrocytes in children: insulin resistance in obese children.

This aim of this study was to determine whether RBC insulin receptor assay represents a clinically useful way of assessing insulin sensitivity in obese children. Steady state plasma glucose (SSPG) was established by a constant infusion of glucose (6 mg/kg/min), insulin (0.8 mU/kg/min) and somatostatin (125 micrograms/m2/h), following the loading dose of somatostatin (125 micrograms/m2). Insulin binding to RBCs was measured by a modified method of Gambhir and was compared with SSPG. Of 21 children with various relative body weight, 8 hyperinsulinemic obese children had a decreased insulin binding to RBCs due to decreased receptor concentrations. The insulin binding was inversely correlated with the fasting serum insulin level and with the insulin area under the O-GTT insulin response curve. In 11 children with various relative body weight, a highly significant inverse relationship was found between SSPG and insulin binding. SSPG was also correlated with the fasting serum insulin level. It was concluded that RBC insulin receptor may quantitatively reflect insulin resistance in obese children, and may be a useful tool for clinical evaluation of tissue insulin sensitivity in children.

In vivo insulin sensitivity and insulin binding to erythrocytes in children

The aim of this study was to determine whether RBC insulin receptor assay represents an useful means for assessment of the clinical sensitivity of insulin. Steady State Plasma Glucose(SSPG) was established by constant infusion of somatostatin (125μg loading followed by 125μg/m2/hr), glucose(6mg/kg/min) and insulin(0.8 mU/kg/min). Insulin binding to RBC receptors was measured and compared with SSPG. Insulin binding to RBC was assayed by the modified method of Gambhir. Plasma insulin area was measured by GTT. A decreased specific insulin binding(SB) to RBC, due to reduced receptor concentrations was observed in hyperinsulinemic obese children. In 21 children with various body weights, SB was inversely related to fasting plasma insulin level (r=-0.651, p<0.01) and also with plasma insulin area (r=-0.547, p<0.01). A highly significant inversed correlation was noted between SSPG and SB(r=-0.807, p<0.005). SSPG was, also, correlated with the fasting plasma insulin level(r=0.669, p<0.05). It was concluded that RBC insulin receptor is an useful tool for clinical evaluation of tissue insulin sensitivity.

Insulin receptor defect in diabetic man with chronic renal failure: A comparison of erythrocyte insulin binding in diabetic and nondiabetic patients on maintenance hemodialysis

125I-Insulin binding to most accessible and easily obtained circulating erythrocytes in 7 diabetic and 16 nondiabetic, nonobese patients with chronic renal failure (CRF) on maintenance hemodialysis was studied and compared with that of the 30 normal, nonobese volunteers. The percent-age of 125I-insulin binding was 4.7 ± 1.8 (mean ± SD) in the diabetic and 13.1 ± 2.4 in the nondiabetic patients with CRF while in the normal subjects it was 9.9 ± 1.4. There was no statistical difference in the fasting levels of glucose, insulin, and glycosylated hemoglobins in all the subjects studied. No correlation ( r = −0.32) between insulin binding and endogenous insulin was observed in these subjects. The diabetic patients with CRF had no circulating factor(s) that would reduce insulin binding to normal erythrocytes. In comparison to the normal subjects, the reduction in insulin binding in the diabetic patients with CRF was accompanied by 47% reduction in insulin receptor sites ( R 0) but with no change in insulin receptor affinity, ( K ̄ e , 0.46 × 10 8 m −1), while in the nondiabetic patients with CRF no change in insulin receptor sites ( R 0) of increased affinity ( K ̄ e , 0.63 × 10 8 m −1) was observed. These findings suggest that insulin binding utilizing the much simpler erythrocyte insulin receptor assay can be used to evaluate alteration in insulin receptor status in CRF. These investigations also provide a very useful assay and characteristic studies for insulin receptors, if receptor modulation acquires a status of treatment for diabetes.

Cell-surface immunoglobulin and insulin receptor expression in an EBV-negative lymphoma cell line and its EBV-converted sublines.

Membrane Ig and insulin receptors were assayed in the EBV-negative Ramos lymphoma line and its EBV-converted sublines by surface fluorescence. 125I-protein A-binding assay, immunoprecipitation, and insulin receptor assay. The original Ramos line expressed surface IgM but not IgD and had a low concentration of insulin receptors. Ten of 10 converted lines expressed IgD and a variable but usually high insulin-binding capacity. Molecular weight analysis of immunoprecipitated Ig showed the presence of 2 mu-chains and 2 delta-chains. One of each heavy chain type could be characterized as membrane Ig, whereas the other was cytoplasmic. Antisera against the lambda-chain precipitated mu and delta heavy chains as well, indicating that the delta-chain expressed on the membrane of the EBV-converted Ramos lines is bound to the lambda light chain. The results show that the differentiation of a Burkitt lymphoma line is not completely "frozen" and can be induced to change in a more "activated" direction by EBV conversion.

Virus-induced decrease of insulin receptors in cultured human cells.

Viral infections may produce abnormalities in carbohydrate metabolism in normal subjects and profound changes in glucose homeostasis in insulin-dependent diabetics. Using an in vitro radio-receptor assay with 125I-labeled insulin and human-amnion (WISH) cells, the effect of viral infections on insulin receptors was examined. Both herpes simplex virus and vesicular stomatitis virus produced a 50% decrease in insulin binding. There was no evidence that this decrease was due to degradation of insulin. On quantitative analysis, this decrease in binding was found to be the result of a decrease in receptor concentration with no change in receptor affinity. The decrease in receptors occurred between 4 and 12 h, at the time viral antigens were being inserted into the plasma membrane of infected cells. Because the t 1/2 of insulin receptors in uninfected cells was between 14 and 24 h, the decrease in insulin receptors cannot be explained solely by virus-induced shut-off of macromolecular synthesis. Moreover, viruses such as encephalomyocarditis that do not insert new antigens into the plasma membrane, did not cause changes in the number of insulin receptors. The most likely explanation is that virus-induced changes in the plasma membrane altered or displaced insulin receptors. It is concluded that the insulin receptor assay is a sensitive and quantitative method for studying the effect of viral infections on cell membranes. These data also suggest that abnormalities in glucose metabolism associated with some viral infections may be due, in part, to changes in the concentration of insulin receptors.

Evaluation of the method of insulin binding studies in human erythrocytes.

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.

Insulin receptor assay in human erythrocytes as an index to insulin sensitivity of body tissues.

Insulin receptor assay in human erythrocytes as an index to insulin sensitivity of body tissues.