Insulin Radioreceptor Assay Research Articles

Preliminary investigations of bony fish--tench (Tinca tinca L.)--erythrocyte insulin receptors.

Specific insulin receptors were identified on tench (Tinca tinca L.) erythrocytes. The samples of blood from 3-4 fish per experiment were pooled in summer. Erythrocytes were washed twice with saline solution, then centrifuged and resuspended in the assay buffer. Incubations were carried out in 0.5 ml samples containing 10(9) cells, with addition of 0.03 nmol/l of 125I-pork insulin in the absence or presence of unlabelled pork or salmon insulin (0.05-2000 nmol/l) for 24 h at 4 degrees C. Calculated from seven experiments specific binding of 125I-insulin was 6.2 +/- 0.2% (mean +/- SD). Scatchard analysis of the binding data from insulin radioreceptor assay showed two different binding sites with high (HAIR) and low (LAIR) affinity. KD values were 0.82 +/- 0.17 nmol/l and 2.05 +/- 1.62 mumol/l, and maximal binding capacity (Bmax) 24.9 +/- 1.6 fmol/10(9) cells and 17.5 +/- 12.2 pmol/10(9) cells, respectively (N = 4). If compared with pork insulin, six to seven times more of salmon insulin was required to displace 50% of iodine labelled hormone. Thus, insulin receptors of tench erythrocytes are similar to those described in other species of fish and other lower vertebrates.

Structurally abnormal insulin in a diabetic patient. Characterization of the mutant insulin A3 (Val----Leu) isolated from the pancreas.

We have recently identified a diabetic patient with marked fasting hyperinsulinemia. Family study revealed that the abnormality was an autosomal dominant trait. High-performance liquid chromatography (HPLC) profile of the patient's serum insulin showed that she had an abnormal insulin in addition to a normal insulin. We have purified her insulin(s) from the specimen of her pancreas, which was biopsied during an operation of cholelithiasis. Insulin was also immunologically purified from the serum of her portal vein. The reverse-phase HPLC analysis revealed that the ratios of normal to abnormal insulin in the pancreas, portal vein, and peripheral vein were 5:4, 4:5, and 1:7, respectively. Radioreceptor assay for insulin using guinea pig kidney membrane revealed that the binding activities of the normal component insulin, the abnormal component insulin and her pancreatic insulin containing both components were 100, 5, and 50% of standard human insulin, respectively. The biological activities of the normal component, the abnormal component and her pancreatic insulin to stimulate glucose oxidation in rat adipocytes were found to be 100, 8, and 60% of standard human insulin, respectively. Analysis of amino acid sequences of the abnormal insulin purified from her pancreas strongly suggested the substitution of leucine for valine at the third position of the A chain, A3 (Val----Leu).

Connective tissue activation. XXVIII. A connective tissue activating peptide from human urine.

A protein factor in human urine which has the ability to activate connective tissue cells has been identified and partially purified; it appears to be different from epidermal growth factor and IgG. This urinary connective tissue activating factor (CTAP-U) is nondialyzable, labile to protease, stable to thiols, heat, and acid, and has an acidic isoelectric point. Purified preparations of CTAP-U have biologic activities that cause human connective tissue cells to synthesize incremental amounts of 14C-hyaluronic acid, 35S-proteoglycans, and 3H-DNA in vitro. The cell spectrum responsive to this substance includes human synovial cells, human chondrocytes, and skin fibroblasts. CTAP-U does not react with antisera to connective tissue activating peptide-III or to antibodies against IgG or its Fc and Fab fragments. Furthermore, CTAP-U does not cross-react in a radioreceptor assay for insulin, basic somatomedin, or epidermal growth factor-urogastrone. Utilizing standardized isolation conditions, CTAP-U preparations with these properties have been isolated from the urine of 6 normal individuals.

New powerful insulin-like protein from human promyelocytic leukemia cells.

Potent insulin-like activity was found in the conditioned medium of human promyelocytic leukemia (HL-60) cells. The conditioned medium of HL-60 cells at high density stimulated [3H]glucose incorporation into lipids in rat adipocytes in a time- and dose-dependent manner. The dose-response curve for this factor was not parallel to that for insulin, and the maximal effect achieved was much greater than reached by insulin or multiplication-stimulating activity. Moreover, the maximal effect reached by either insulin or the conditioned medium was additive. The insulin-like activity was not suppressed in the presence of antiinsulin antibody. Insulin-like activity was not detectable by radioreceptor assay for insulin, suggesting that the factor does not act through the insulin receptor. The factor in the conditioned medium of HL-60 cells was heat stable and sensitive to trypsin. When the conditioned medium was subjected to gel filtration on a Sephadex G-100 column, the major part of insulin-like activity eluted in the position corresponding to an apparent molecular weight between RNAase and insulin markers. The remaining activity, approximately 10% of the total, appeared with a larger molecular weight species. On isoelectric focusing of the smaller molecular species, insulin-like activity was largely focused in the position corresponding to pI 7.8-8.2.

Quality control of insulin radioreceptor assay for human erythrocytes. Effect of ageing of mono-125I-Tyr-A14-insulin preparation.

The quality control of insulin radioreceptor assay for human erythrocytes is based on the storage of erythrocyte preparations in Hepes buffer of pH 8.0, containing 10 g/l of albumin and 20 mmol/l of glucose. The change of erythrocytes into spherocytes and crenated cells reduces the apparent number of insulin receptors in a relatively constant way by less than 8% a week after 10 days of storage. At the same time the dissociation constants of the insulin-receptor complex increase rapidly. Thus the use of a preparation must be limited to controlling the determination of the insulin binding sites of erythrocytes, and not to the measurement of the affinities of the receptors. When mono-125I-Tyr-A14-insulin gets old, a slow decrease in the insulin binding sites can be measured, but the dissociation constants of the insulin receptor complex are not affected.

Insulin radioreceptor assay on murine splenic leukocytes and peripheral erythrocytes.

Insulin radioreceptor assays were developed using splenic leukocytes and peripheral erythrocytes from individual mice. Splenic leukocytes were prepared using an NH4Cl buffer which did not alter insulin binding, but gave much higher yields than density gradient methods. Mouse erythrocytes were isolated from heparinized blood by three passages over a Boyum gradient, and a similar buffer was used to separate cells from free [125I]iodoinsulin at the end of the binding incubation. Insulin binding to both splenic leukocytes and peripheral erythrocytes had typical pH, temperature, and time dependencies, and increased linearly with an increased number of cells. Optimal conditions for the splenic leukocytes (6 X 10(7)/ml) consisted of incubation with [125I]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.0. In cells from 20 individual mice, the specific [125I]iodoinsulin binding was 2.6 +/- 0.1% (SEM), and nonspecific binding was 0.3 +/- 0.04% (10.6% of total binding). Erythrocytes (2.8 X 10(9)/ml) were incubated with [125I]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.2. In cells from 25 individual mice, the specific [125I]iodoinsulin binding was 4.5 +/- 0.2%, and nonspecific binding was 0.7 +/- 0.03% (13.6% of total binding). In both splenic leukocytes and peripheral erythrocytes, analysis of equilibrium binding data produced curvilinear Scatchard plots with approximately 3500 binding sites/leukocyte and 20 binding sites/erythrocyte. These data demonstrate that adequate numbers of splenic leukocytes and peripheral erythrocytes can be obtained from individual mice to study insulin binding in a precise and reproducible manner. This should facilitate direct comparisons of these cells with classical target cells in a variety of mouse model diseases.

Insulin degradation by human erythrocyte lysates.

In vitro hemolysates of isolated human erythrocytes degrade 125I-labeled insulin. Ten- to 100-fold dilutions of the hemolysate give a proportionately decreased degradation of 125I-labeled insulin at 37 degrees C, while dilutions of up to eightfold do not. Like the control, diluted "Buffer G" containing 5 mmol/L Tris and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer alone, more than 500-fold dilutions of the hemolysate or boiled hemolysate (in buffer) caused negligible (less than 1%) degradation of the labeled insulin. We conclude that accurate insulin-binding data during erythrocyte insulin radioreceptor assay under optimum conditions (Clin. Chem. 23: 1590-1595, 1977) depend on avoiding or minimizing hemolysis.

Basic peptide with insulin-like activity in human serum.

A peptide with insulin-like activity was isolated from human plasma. In the purification, insulin-like activity (ILA) was monitored by radioreceptor assay for insulin, using human placental membrane and [125I]-insulin. ILA was extracted from Cohn fraction III with acid-ethanol and chromatographed on Sephadex G-75 in 1% formic acid. When the active fractions were subjected to ion-exchange chromatography with CM-cellulose, the ILA was adsorbed to the column at pH 5.0 and was eluted with a gradient of ammonium acetate. The chromatographic behavior of the ILA was not identical to that of somatomedin A as determined by radioreceptor assay (RRA) for the latter. On isoelectric focusing of the ILA from the CM-cellulose column, insulin-like activity was distributed over a wide pH range. The ILA that was focused at pH 7.5-9.0 was further purified by gel filtration on Sephadex G-50 in 1 M acetic acid. The specific activity of the basic ILA was approximately 200 mU insulin equivalent /mg protein. The apparent molecular weight of the material was estimated to be 7,000. It stimulated [14C]-glucose oxidation in rat epididymal fat cells and had sulfation activity in chick chondrocytes. Furthermore, the basic ILA had a potent mitogenic activity in Balb/c-3T3 cells. Thus, the basic ILA is qualified as an 'insulin-like growth factor' (IGF). It is obviously different from somatomedin A, and may be closely related to IGF-I or somatomedin C. Subsequently, RRA for the basic ILA was developed in serum concentration of the basic ILA were determined. The serum concentrations of the basic ILA were high in acromegalics and low in patients with hypopituitarism. Thus, the basic ILA is entitled to be one of the IGFs.

Radioreceptor assay of insulin using rabbit erythrocyte membrane.

A simple radioreceptor assay for insulin was established with sensitivity sufficient to detect 10 μU/ml of insulin in human sera. This radioreceptor assay was carried out at 0° using rabbit erythrocyte membranes. The binding assay for the standard curve was performed in the presence of insulin-free serum, since the binding of insulin was greatly reduced by the addition of serum. Under the conditions used, 38% of the initial binding of 125I-insulin was displaced by 250μU/ml of native insulin. The sensitivity of the radioreceptor assay was dependent on the concentrations of both membrane protein and 125I-insulin. The ratio of IRI to insulin values obtained by the present assay was 0.93±0.10 for 19 healthy and diabetic subjects.

Retroperitoneal hemangiopericytoma associated with hypoglycemia: Report of a case

A 34-year-old woman had episodes of hypoglycemic attack 8 years after a surgical resection of a retroperitoneal hemangiopericytoma. In spite of normal levels of serum IRI, insulin radioreceptor assay demonstrated high level of plasma ILA (insulin like activity). The patient underwent resection of a recurrent retroperitoneal tumor with metastatic lesions of the liver. Postoperatively, ILA level in plasma by insulin radioreceptor assay decreased, and hypoglycemic attacks disappeared. Therefore, this associated hypoglycemia was presumed to be not caused by excess glucose consumption by the tumor, not by excess secretion of IRI by the tumor, but caused by the presence of high level of ILA related to the tumor.

Erythrocytes: A New Cell Type for the Evaluation of Insulin Receptor Defects in Diabetic Humans

Human erythrocytes have specific insulin receptors. When studied in an insulin radioreceptor assay, erythrocytes from adult-onset, nonobese diabetic subjects bound at least 42 percent less insulin than the normal subjects at insulin concentrations from 0.1 to 100 nanograms per milliliter. The diabetic subjects had 190 insulin receptor sites per cell as compared with the 380 insulin receptor sites per cell for the normal subjects. The deficit of insulin binding in the diabetic subject was thus associated with a fewer number of insulin binding sites per cell with little or no change in affinity. The erythrocyte is a readily available cell for the evaluation of cellular insulin receptor activity.

Partial purification and characterization of a binding protein for insulin-like activity (ILAs) in human amniotic fluid: a possible inhibitor of insulin-like activity.

An insulin radioreceptor assay (INS-RRA) and an insulin-like activity radioreceptor assay (ILAs-RRA) have been utilized to partially purify and characterize a protein from human amniotic fluid with ILAs-RRA reactivity. An acid-ethanol soluble protein with an apparent molecular weight of 34,500 daltons by calibrated Sephadex chromatography and an isoelectric point (pI) of 4.7 accounts for all of the ILA'S-RRA reactivity present in human amniotic fluid. Since this protein has been found to be a binding protein for ILAs, but not insulin, it has been termed amniotic fluid binding protein or AFBP. AFBP is reactive in a non-parallel manner in the ILAs-RRA and totally inactive in the INS-RRA. The activity of AFBP in the ILAs-RRA is thus to the competition of AFBP with the placental membrane receptor for the [125I]ILAs tracer employed in the ILA'S-RRA. AFBP inhibits the activity of employed insulin, in the INS-RRA, presumably by binding ILAs, while being inactive itself. In two biological assays studied to date, the rat epididymal fat pad assay and the rabbit chondrocyte sulphation assay, AFBP also inhibits the activity of added ILAs. These observations raise the possibility that binding protein(s) for insulin-like peptides may function as inhibitors of their bioactivity in different physiologic and pathologic states. The relation of AFBP to binding protein(s) in human plasma remains to be clarified.

Partial Purification, Characterization, and Assay of a Slightly Acidic Insulin-Like Peptide (ILAs) from Human Plasma*

An insulin radioreceptor assay (RRA) using human placental microsomal membranes was used to measure insulin-like activity (ILA) extracted from human plasma concentrates (Cohn fraction IV-4) by acid ethanol. The soluble activity (ILAs), chromatographed on Sephadex G-75 in 1 M acetic acid, migrated as a small molecule (fractional elution volume, 0.56) ahead of insulin (fractional elution volume, 0.70), whereas at neutral pH, ILAs migrated as a large molecular weight species. The ILAs peak from acid gel filtration on Sephadex was further purified by chromatography on carboxymethyl cellulose (CMC). The ILAs peak from both Sephadex and CMC diluted parallel to the porcine insulin standard in the insulin RRA and was totally unreactive in an insulin RIA. The CMC-purified material was iodinated and purified by binding to and elution from human placental membranes. The binding of [125I]ILAs to human placental membranes was inhibited only minimally by insulin and proinsulin and not at all by epidermal growth factor, nerve growth factor, glucagon, or lactogenic hormones, including human growth hormone. Multiplication-stimulating activity (MSA) inhibited in a manner parallel to ILAs. A Scatchard plot of the binding data was nonlinear. Sephadex ILAs was subjected to isoelectric focusing. The fractions assayed in both insulin and ILAs RRAs yielded comparable results. Peaks of ILA were observed at pHs 5.3, 6.6, and 8.4. When CMC-ILA was subjected to isoelectric focusing in polyacrylamide, a single peak of activity migrating between pH 6.2-6.8 was seen. [125I]ILAs focused at exactly the same pH. Electrophoresis of CMC-ILAs in acid-urea revealed a sharp peak of activity migrating with one of the five protein bands seen after staining. Again, [125I]ILAs comigrated with unlabeled ILAs. The molecular weight of ILAs, as determined on a calibrated Sephadex G-150 column at neutral pH, was 9,000-10,000 daltons. CMC-ILAs stimulated [14C]glucose incorporation into triglycerides of rat adipose tissue and augmented [3H]thymidine incorporation into human fibroblasts, chicken embryo fibroblasts, and BALB 3T3 cells as well as [35S]sulfate incorporation into macromolecules of rabbit chondrocyte culture medium. In summary, ILAs isolated on the basis of a RRA for insulin is a slightly acidic peptide with some of the biological activities expected of a somatomedin.

Insulin, insulin-like activity and the human placenta

Specific insulin binding sites can be demonstrated in human placental membranes. Binding is readily reversible. Both the rates of binding and dissociation are temperature sensitive. Insulin analogues bind with affinities which parallel their biological activity. The affinity for insulin is of sufficient magnitude to bind physiologic levels of insulin. Insulin eluted from these binding sites is structurally intact. The binding sites are partially protein in composition and are found in the placental tissue of many species. The binding sites have the properties of biologically significant receptors in well-defined insulin target tissues. This suggests a direct role for insulin in regulating placental function. Two different soluble insulin degrading enzymes have been described in human placenta. In addition, a third membrane-associated degrading activity can be demonstrated. This enzyme was partially washed out of the membranes under circumstances which did not influence the insulin binding sites. This dissociation of degrading activity and binding suggests that they are not intimately related. An insulin radioreceptor assay (RRA) was used to follow the purification of an insulin-like peptide (ILAs, 7–12 × 10 3 daltons) from human Cohn fraction IV-4. ILAs had chemical and biological properties like those of somatomedin and NSILAs. It bound in a specific manner to a unique human placental receptor suggesting a role for it in placental function. Using this receptor and [ 125I]-ILAs, an ILAs RRA was developed and validated. The measurement of ILA in rat serum with the ILAs RRA revealed a large molecular weight crossreacting material whose level was greatly reduced by hypophysectomy. It has not been excluded that this material crossreacts in our assay through an ability to bind [ 125I]-ILAs. Further study must be aimed at characterizing the nature of insulin-like peptides in the circulation of animals and man.

Characterization of a Protein in Mid-Term Human Amniotic Fluid which Reacts in the Somatomedin-C Radioreceptor Assay*

Amniotic fluid contains materials other than insulin which react in a somatomedin C radioreceptor assay using human placental membranes. The material in mid-gestational amniotic fluid which reacted with the somatomedin C radioreceptor assay eluted slightly after albumin from a Sephadex G-150 column equilibrated with 0.1M NH4HCO3. Neither boiling nor treatment of this fraction with 1% formic acid yielded small molecular weight somatomedin-like peptides. Separation of the somatomedin reactive material (Sm RM) from albumin was achieved by gel filtration through Ultrogel AcA44 in 3.1M NH4HCO3. The active product had an apparent molecular weight of 33,000 to 35,000 Daltons; its isoelectric point determined by focusing was between 4.1 and 5.1. The purified amniotic fluid protein displaced somatomedin C in the somatomedin C radioreceptor assay but did not compete with insulin in the insulin radioreceptor assay. Sm RM produced only a slight stimulation of thymidine uptake in human fibroblasts but was inactive in stimulating sulfate uptake in hypox rat costal cartilage. In human fibroblast cultures Sm RM inhibited the stimulation of thymidine uptake induced by human serum and by purified rat somatomedin. When Sm RM was added to the 125I somatomedin C, some of the radioactivity eluted from gel filtration at pH 8.6 was converted to a molecular weight complex of about 43,000. We conclude that the material which we have isolated from mid-gestational amniotic fluid is a protein which may bind somatomedin and make it unavailable to the somatomedin receptor of human placenta and human fibroblasts.