Biomedical subjects
Publications and source records attributed to A McElduff.
Explore the source record for details and available documents.
We have identified and characterized insulin receptors on Chinese hamster ovary (CHO) cells. Insulin binds in a time, temperature and pH dependent fashion and insulin analogues compete for 125I-insulin binding in order of their biological potencies. Furthermore, two CHO cell glycosylation mutants, B4-2-1, lacking high mannose containing glycoproteins, and Lec 1.3c, lacking complex carbohydrate containing glycoproteins, bind insulin with a much higher and lower affinity respectively than wild type cells. This is the first report of insulin receptors on CHO cells and the first to use glycosylation mutants to study the effects of abnormal carbohydrates on insulin binding.
We have examined insulin binding to the erythrocyte insulin receptor in normal males following a short fast and a test meal or glucose load. The binding increased following the fast. This increase appeared to be due to increased receptor affinity. Following the meal or glucose load no change in insulin binding was detected. These results are compared with conflicting data in the literature and possible technical explanations for the discrepancies are explored.
Previous studies of the insulin receptor in disease states have utilized primarily techniques of equilibrium binding and, to a limited extent structural, analysis. Though techniques have been developed to study receptor degradation in normal cells, they have not been applied to disease states. In the present study we have examined insulin receptor degradation rate in B lymphocytes that were obtained from peripheral blood of normal subjects and patients with several syndromes of extreme insulin resistance. B lymphocytes were established in culture from each patient's peripheral cells by transformation with Epstein-Barr virus. The insulin receptors were surface labeled using Na125I/lactoperoxidase and the cells were returned to incubate in growth media. After varying periods of incubation, aliquots of cells were solubilized and the cell content of labeled receptor subunits were measured by immunoprecipitation with anti-receptor antibodies and NaDodSO4/polyacrylamide gel electrophoresis. The fall in 125I-insulin receptor content approximated a single exponential and was quantitated as receptor subunit half-life (t1/2). In cell lines from four patients in whom the number of insulin receptors was reduced by greater than 90%, the rate of receptor loss was greater than normal (t1/2 equals 3.8 +/- 0.9 h vs. 6.5 +/- 1.2 h; mean +/- SD, P less than 0.01). However, a similar acceleration in receptor degradation was seen in cells from five patients with extreme insulin resistance but low-normal insulin receptor concentration (t1/2 equals 4.4 +/- 0.9 h). This group included cells from one patient with a qualitatively abnormal receptor. Thus, all the patients with genetic syndromes of insulin resistance had accelerated receptor degradation, regardless of their receptor concentration. By contrast, insulin receptors on cultured lymphocytes that were obtained from patients with extreme insulin resistance secondary to autoantibodies to the insulin receptor had normal receptor degradation (t1/2 equals 6.1 +/- 1.9 h). We conclude that (a) accelerated insulin receptor degradation is an additional feature of cells from patients with genetic forms of insulin resistance; (b) that accelerated insulin receptor degradation may explain the low-normal receptor concentrations that were seen in some patients with extreme insulin resistance; and (c) that accelerated degradation does not explain the decreased receptor concentration in patients with very low insulin receptor binding and, therefore, by inference, a defect in receptor synthesis must be present in this subgroup.
Binding of insulin to its receptor followed by covalent cross-linking with disuccinimidyl suberate (DSS) dramatically impairs the ability of antiinsulin antibodies (both polyclonal and monoclonal) to bind to the insulin moiety. We have used dithiotheitol, which has major effects on the oligomeric structure of the insulin receptor, to determine if this decreased antibody recognition is due to alteration in the conformation of insulin itself or to steric factors. Treatment of the covalently cross-linked insulin-receptor complex with dithiothreitol (DTT) increased the ability of the polyclonal and two monoclonal antiinsulin antibodies to immunoprecipitate the insulin-receptor complex. This treatment decreased immunoprecipitation by antireceptor antibodies. The effect of DTT may have been due to a reversal of either a binding-induced conformational change in the insulin moiety or an alteration in the conformation of the insulin-receptor complex, thereby decreasing steric hindrance. In an effort to choose between these two possible explanations, we prepared a biotinylated derivative of insulin which was cross-linked to the receptor. Since the biotin moiety is relatively rigid, it seemed improbable that binding to the receptor would alter the conformation of the epitope recognized by antibiotin antibodies and that the change would be reversed by DTT. Treatment of the cross-linked biotin-insulin-receptor complex with DTT did increase the ability of both antiinsulin and antibiotin antibodies to immunoprecipitate the cross-linked receptor complex. Identification of the cross-linked receptor on reduced sodium dodecyl sulfate-polyacrylamide gels confirms that the DTT treatment alters the distribution of the oligomeric forms of the receptor. These studies favor the hypothesis that when bound to its receptor, most of the insulin molecule is sequestered within the receptor-binding site such that there is steric hindrance to the approach of antiinsulin antibodies. Moreover, DTT alters the conformation of the cross-linked insulin-receptor complex so as to decrease this steric hindrance.
Levels of fasting plasma insulin are generally inversely correlated with 125I-insulin binding to circulating blood cells. In disease states associated with hyperinsulinemia (e.g., obesity and non-insulin-dependent diabetes mellitus), 125I-insulin binding is usually low. In contrast, 125I-insulin binding to circulating cells may be normal in patients with certain forms of extreme insulin resistance despite marked hyperinsulinemia. To explain this paradox, it has been proposed that postbinding defects in insulin action may give rise to defects in downregulation. We have employed cultured Epstein-Barr virus (EBV)-transformed lymphocytes from eight patients with extreme insulin resistance to address the question of whether there is a defect in the downregulation process in vitro. In this cell type, insulin leads to a decrease in the number of insulin receptors on the cell surface by accelerating the rate of degradation of insulin receptors. We could not detect any abnormality in in vitro down-regulation with cultured EBV-transformed lymphocytes from insulin-resistant patients. The apparent discrepancy between the in vivo and in vitro studies raises the possibility that some factor in the patient's internal milieu may prevent insulin-induced downregulation. An alternative possible explanation might be that the mechanism of downregulation in vitro differs from the mechanism whereby receptor number is regulated in vivo in insulin's target cells.
The interaction between insulin and its receptor was investigated using both monoclonal and polyclonal anti-insulin antibodies. After covalent cross-linking of 125I-insulin to the insulin receptor on cultured human lymphocytes (IM-9 cells) using disuccinimidyl suberate, we inquired whether the insulin-receptor complex could be immunoprecipitated with anti-insulin antibodies. While a polyclonal guinea pig anti-insulin antiserum succeeded in immunoprecipitating receptor-bound 125I-insulin, binding to the receptor decreased the avidity of the antiserum for the insulin moiety by a factor of approximately 1000-fold. Sixteen distinct monoclonal murine anti-insulin antibodies were employed to immunoprecipitate receptor-bound 125I-insulin. Of these 16 monoclonal antibodies, only one (antibody 5.9F4) could be shown to recognize receptor-bound 125I-insulin. Moreover, even with antibody 5.9F4, binding of 125I-insulin to its receptor reduced the affinity of the antibody by a factor of 10- to 100-fold. These data strongly suggest that, when insulin binds to its receptor, the majority of the insulin molecule is unavailable for binding by anti-insulin antibodies. It seems likely that the hormone binding site on the receptor may be very large, thereby allowing for sequestration of the majority of the insulin molecule with relatively little of the hormone remaining exposed.
Explore the source record for details and available documents.
A precise, reproducible radioreceptor assay has been developed for insulin receptors on human erythrocytes. The mean specific binding in 11 normal volunteers was 7.7% +/- 1.6 (S.D.) per 2.25 x 10(9) erythrocytes. The intra and inter assay coefficients of variation are 4%. The erythrocyte insulin receptor is specific for insulin and recognises insulin analogues in proportion to their respective biological activities. The pH and temperature dependence, the negative cooperativity and the inverse relationship between specific binding and fasting plasma insulin levels suggest it is similar to insulin receptors elsewhere. The erythrocyte insulin receptor offers a model for the study of insulin/insulin receptor interaction in man.
In vitro incubation of human erythrocytes with disulfide reducing agents (dithiothreitol and 2-mercaptoethanol) produces a significant increase in specific binding of 125I insulin to the insulin receptor. Insulin binding is maximal in the presence of 10(-4)M dithiothreitol and declines abruptly at higher concentrations. Preincubation of red cells with these agents and repeated washing prior to inclusion in the receptor assay produced similar effects, but at higher concentrations of reducing agent (10(-2)M dithiothreitol). The increased binding of insulin was consistent with an increase in receptor affinity induced by the reducing agents. Agents which alkylate or oxidise free sulfhydryl groups tended to decrease specific binding of 125I insulin to the receptor. These data suggest that alterations of sulfhydryl groups may be an important mechanism in modulating insulin receptor affinity.
A 16-year-old girl presented with severe, prolonged insulin resistance. Insulin antibodies, initially thought to be responsible for the insulin resistance, were suppressed using monocomponent insulin and immunosuppressive therapy; however insulin resistance persisted. Insulin kinetic studies suggested abnormal metabolism of a bolus injection of 125I insulin and the reappearance in the circulation of radioactive products, demonstrated by chromatography to be of different molecular weight to insulin. These products were of similar molecular weight to material obtained by incubating 125I insulin with protease. Trasylol significantly reduced the patient's insulin requirements and normalised the disappearance of 125I insulin from the circulation. Prolonged treatment with Trasylol resulted in a fall in insulin requirement to non "insulin-resistance" levels. The insulin requirement remained static when Trasylol was ceased. We propose abnormally rapid insulin degradation to be a new mechanism of resistance to insulin therapy.
A patient with metastatic medullary carcinoma of the thyroid, Cushing's syndrome, and severe diarrhoea is presented. The manifestations of Cushing's syndrome were controlled with a combination of aminogluthethimide and metyrapone. The cause of the diarrhoea has not been found, and it remains resistant to treatment.
Iliac crest biopsies and serum specimens were obtained from 36 non-dialyzed uraemic patients. The mean serum 25-hydroxycalciferol concentration of the patients was lower than that of normal subjects and a significant correlation was found between 25-hydroxycalciferol values and the severity of osteomalacia. Parathyroid osteopathy was significantly correlated with serum immunoreactive parathyroid hormone but not with serum 25-hydroxycalciferol values. While normal serum 25-hydroxycalciferol concentrations in uraemic patients do not exclude the presence of osteomalacia, a low concentration is virtually diagnostic of this disorder.
Mean 24-hour integrated concentrations of serum prolactin (PRL) in 8 breast-feeding mothers (62 +/- 10.4 microgram/l) was significantly higher than in 4 control subjects (14.8 +/- 3.3 microgram/l). No correlation was found between the mean integrated concentrations and time after delivery or breast-feeding status. There was no relationship between the mean integrated concentration of PRL or the basal PRL and the frequency of feedings, the total suckling time, the mean interval between feedings or the longest interval between feedings in the 24-hour period. The study provides conclusive evidence for increased prolactin secretion in long-term puerperal lactation.
We estimated the glomerular filtration rate in 33 patients from our diabetic clinic using three methods: the creatinine clearance measured from a timed urine sample and a serum creatinine; the creatinine clearance calculated from the serum creatinine according to the formula of Cockcroft and Gault; and, the plasma clearance of ethylenediaminetetra-acetic acid (EDTA) labeled with 51Cr ([51Cr]EDTA). We repeated the measurements in seven subjects. The measured creatinine clearance was not reproducible. The other two methods were correlated, but not according to the formula y = x. The calculated creatinine clearance significantly underestimated the [51Cr]EDTA clearance particularly at higher [51Cr]EDTA clearance rates. [51Cr]EDTA clearance has been shown by others to parallel, but underestimate, inulin clearance, the optimal method of estimating glomerular filtration rate but difficult to perform in routine practice. Accurately measuring renal function in routine clinical practice is difficult, and this must be borne in mind when making clinical decisions based on current measurements.
We have performed a cross-sectional analysis of the relationship between prorenin values and the microvascular complications of diabetes in a well controlled population of insulin-dependent diabetes mellitus (IDDM) subjects. One hundred and thirty-nine subjects (75 men, 64 women, age 44 +/- 17 years; duration of diabetes 19 +/- 15 years), formed the study group. Sixty-seven subjects (48.2%) had no complications, 55 (39.6%) had retinopathy alone, and 17 (12.2%) had retinopathy and albuminuria. Patients with no complications had lower prorenin values than those with microvascular complications (p < 0.001), whilst patients with both albuminuria and retinopathy had higher values than those with retinopathy alone (p < 0.05). Retinopathy was associated with duration of diabetes (p < 0.0001), diastolic blood pressure (p < 0.02) and albuminuria (p < 0.0001) while albuminuria was associated with prorenin (p < 0.02), serum triglyceride (p < 0.01) and retinopathy (p < 0.001). Patients with albuminuria were 5.5 times more likely to have raised prorenin values (>80 ng/mL/h) than those with normal albumin excretion [95% confidence interval (CI): 1.48-20.12] and those with retinopathy alone were 2.5 times as likely (95% CI: 1.19-5.15). Eighty patients with IDDM (40 males, 40 females; age: 47 +/- 17 years; duration of diabetes: 20 +/- 15 years), had retinal photography performed to determine the association between the severity of retinopathy and prorenin values. Retinopathy was more severe in patients with retinopathy and albuminuria than in those with retinopathy alone (p < 0.002). When the prorenin values of patients with more marked retinopathy (eye grade greater than 3) were compared, prorenin values of those with retinopathy and albuminuria were greater than those of patients with retinopathy alone [269 (139-1406) versus 91 (41-273) ng/mL/h: geometric mean (range); p < 0.05]. Furthermore, when patients without albuminuria were considered, there was no significant difference between the prorenin levels of patients with more severe retinopathy (eye grade >3) when compared to patients with lesser degrees of retinopathy [91 (41-273) versus 69 (23-375). In patients with microvascular complications, prorenin values were independently predicted by albuminuria (p < 0.0001) and diastolic blood pressure (p < 0.02) but not the severity of retinopathy. In conclusion, prorenin values are significantly associated with the presence of microvascular complications in patients with IDDM. The association with albuminuria may be stronger than the association with retinopathy.
Explore the source record for details and available documents.
The ultimate or primary goal of therapy for type 2 diabetes is to prevent the mortality and morbidity related to the microvascular and macrovascular complications. It is increasingly obvious that to achieve this on a global perspective we will need to identify better and more effective strategies to prevent diabetes and its precursors, obesity and inactivity. Similarly, since a large percentage (at least 50%) of patients with type 2 diabetes are undiagnosed, we need to improve the implementation of strategies aimed at detecting this undiagnosed population. Without a diagnosis we will not prevent, detect or treat the complications of diabetes. Finally, therapy of type 2 diabetes is evolving as the evidence base for clinical decision-making grows and as new therapeutic options become available. The therapeutic approach has broadened considerably to include more intensive treatment of the diseases frequently associated with type 2 diabetes and major contributors to morbidity and mortality, namely hypertension and dyslipidaemia.