Showing papers on "Proinsulin published in 1974"
TL;DR: Human placentas have been used to further investigate the binding of insulin and somatomedin to cell membrane receptors to define the optimal conditions for a competitive binding assay for insulin and insulin-like peptides.
Abstract: Human placentas have been used to further investigate the binding of insulin and somatomedin to cell membrane receptors. Conditions for optimal binding of 125I-insulin were defined for both participate membrane preparations and membranes solubilized in Triton X-100. Human placental membranes are a rich source of high affinity insulin receptors. As in previous studies with rat liver and fat cell preparations, only somatomedin and proinsulin were effective competitors for binding to the insulin receptor. These findings were used to define the optimal conditions for a competitive binding assay for insulin and insulin-like peptides. This assay had threshold sensitivities of 10 μU/ml for insulin and less than 0.1 U/ml for somatomedin. Incubation at 4 C for 15–18 hr essentially eliminated proteolytic degradation of the labeled hormone and led to strikingly better binding than was observed at higher temperatures. A highly purified preparation of somatomedin-C labeled with radioactive iodine was found to...
303 citations
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TL;DR: The amino acid sequence of mouse NGF exhibited significant structural relatedness with the family of insulins and proinsulins as mentioned in this paper, which is indicative of distant, but significant evolutionary relatedness.
200 citations
TL;DR: There was considerable species variation in insulin binding, with the placental membranes of the guinea pig and monkey having the highest and those of the rat having the lowest binding.
Abstract: The existence of polypeptide hormone receptors in the human placenta was evaluated by studying the specific binding of 125-I-labeled insulin, human growth hormone (hGH), human pro lac tin (hPRL) and glucagon to a defined placental membrane fraction. Only insulin showed specific binding to placental membranes. The binding of 125-I-insulin was time and temperature dependent. Its dissociation from the membrane was first order with a half time, at 24° C, of twenty minutes. Specific binding was readily observed at a concentration of 5 × 10−11 (7.5 μU./ml.). Inhibition of 125-I-insulin binding by unlabeled insulin was 30 per cent at 10−9 M, and >90 per cent at 10−9 M. Desalanine insulin was equally effective in inhibiting the binding of 125-I-insulin. Proinsulin was about twenty times less effective, and desoctapeptide insulin was even less effective. Structurally unrelated polypeptide hormones were without significant inhibitory effect. Binding sites of relatively high affinity (K1 = 4.2 × 108 M−1) and low capacity could be distinguished from those of lower affinity (K2 = 0.7 × 108M−1) and higher capacity. Insulin degrading activity was shown to be present in the placental membranes. Under standard binding assay conditions, less than 20 per cent of the 125-I-insulin present was degraded. The 125-I-insulin could be eluted from the membranes and appeared intact by several criteria. Specific binding was augmented by Ca++ and other divalent cations but was unaffected by high sodium chloride concentrations. Binding was greatly reduced by pretreatment of membranes with proteolytic enzymes. Incubation with phospholipase C, RNase and neur-aminidase had relatively little effect on binding.
Insulin binding was unaffected by maternal diabetes but was reduced in membranes from early gestational placentas. There was considerable species variation in insulin binding, with the placental membranes of the guinea pig and monkey having the highest and those of the rat having the lowest binding.
The characteristics of the insulin binding sites in the human placenta are similar to those in established insulin target tissues.
165 citations
TL;DR: C-peptide immunoreactivity in plasma of normal subjects assayed by the radioimmunoassay system was 0.88±0.21ng/ml, and proinsulin and Proinsulin-like components had been reported to be very low in plasma.
Abstract: Radioimmunoassay of human C-peptide was established using antiserum to synthetic human connecting peptide and synthetic tyrosylated human connecting peptide. Synthetic human connecting peptide and natural human C-peptide showed the same degree of cross-reactivity with the synthetic human connecting peptide antiserum. Natural human proinsulin reacted approximately one fourth as well as the human connecting peptide or natural human C-peptide when expressed on an equimolar basis.Synthetic porcine and bovine connecting peptide, human insulin and natural porcine proinsulin did not react with the human connecting peptide antiserum. Synthetic human connecting peptide did not cross with insulin antiserum. The senstitivity of the radioimmunoassay was 0.05ng/tube.The assay system was available to determine C-peptide immunoreactivity in plasma, because proinsulin and proinsulin-like components had been reported to be very low in plasma. The fasting level of C-peptide immunoreactivity in plasma of normal subjects assayed by this system was 0.88±0.21ng/ml.
130 citations
TL;DR: The studies demonstrate and characterize the binding of insulin to normal isolated human adipocytes; they indicate that the study of insulin-adipocyte binding is a possible means to gain further insight into the mechanism of altered response to insulin in human disease states.
Abstract: In order to study the binding of insulin to insulin-sensitive human tissue, we isolated adipocytes from surgically obtained human fat tissue. Our data demonstrate that insulin readily and specifically binds to isolated adipocytes. The time course of this reaction indicates that steady state binding conditions occur at forty-five minutes, with a subsequent decline in binding later. There is no appreciable insulin or receptor degradation before forty-five minutes, suggesting a true equilibrium. After forty-five minutes the decline in binding can be accounted for by insulin plus receptor degradation. At 3 × 10 −11 M 125-I-insulin and 2 × 10 5 cells per milliliter, 1.8 to 2.4 per cent of the 125-I-insulin was bound. The specificity of this binding reaction is demonstrated by the high concentrations of thyroid stimulating hormone and human growth hormone that are without effect on binding, while proinsulin and desalanine insulin inhibit binding in proportion to their biologic activity. On the other hand, binding can be readily inhibited by porcine insulin at physiologic concentrations, i.e. binding is 13 per cent inhibited at 1 ng. per milliliter and 50 per cent inhibited at 8.6 ng. per milliliter. The kinetic behavior of this reaction can be approximated by Scatchard analysis, which indicates 50,000 high affinity sites per cell with a dissociation constant (Kd) of 1.9 × 10 −9 M per liter and 250,000 low affinity sites per cell with a Kd of 8 × 10 −9 M per liter. In conclusion, these studies demonstrate and characterize the binding of insulin to normal isolated human adipocytes; they indicate that the study of insulin-adipocyte binding is a possible means to gain further insight into the mechanism of altered response to insulin in human disease states.
115 citations
TL;DR: It appears that, in liver, insulin and NSILA-s bind to two types of receptors with markedly different affinities.
96 citations
TL;DR: Fatty acid and sterol biosynthesis was studied in several cell lines in culture and indicated that the increased fatty acid synthetase activity observed after the removal of serum from the medium or the addition of insulin to serum-free medium was due to increased levels of enzyme in the cells.
80 citations
TL;DR: It is suggested that diseases that affect the B-cell's ability to store insulin or prevent the secretory granule of the B -cell from undergoing full maturation will be associated with an increased secretion from immature granules, that contain a higher percentage of proinsulin-like components.
Abstract: The proportion of total plasma immunoreactive insulin comprising the proinsulin-like component was determined in 5 diabetic subjects manifesting fasting hyperglycemia, severe glucose intolerance, and insulinopenia, and also in one insulinopenic patient both before and after removal of a phaeochromocytoma. The proinsulin-like component comprised a mean of 37% (range 30–50%) of the total immunoreactive insulin in the basal state, 27% (range 20–32%) at 15 min, and 32% (range 23–50%) 120 min after 100g of oral glucose. These values are significantly higher than in normal subjects or patients with hyperinsulinemic responses (excepting patients with insulinoma). Our studies suggest that diseases that affect the B-cell's ability to store insulin or prevent the secretory granule of the B-cell from undergoing full maturation will be associated with an increased secretion from immature granules, that contain a higher percentage of proinsulin-like components. The proportion of the total immunoreactive insulin comprising the proinsulin-like component is, therefore, a reflection of the integrity of the B-cell granule.
74 citations
TL;DR: Results indicate that the lower affinity of proinsulin for the insulin receptor in the liver is an intrinsic property of the Proinsulin molecule, in addition to a slower degradation of the prohormone.
Abstract: The interactions of proinsulin with the insulin-specific receptors were investigated in purified rat liver plasma membranes. These studies were de- signed to characterize the binding of proinsulin to the insulin receptors, to search for proinsulin-specific re- ceptor sites, and to examine the possibility of proinsulin conversion at the insulin receptor site. Proinsulin was only 3-5% as potent as insulin in binding to insulin receptors. Proinsulin reacted with all of the insulin- specific receptors, and direct binding studies of (HI)- porcine proinsulin and (HI)rat proinsulin did not re- veal proinsulin-specific receptor sites other than the insulin receptors in rat liver membranes. Quantitative data derived from steady-state and tran- sient-state comparative binding studies of both ('I)- proinsulin and ("fJ) insulin indicated that a 20-fold lower association rate constant essentially accounts for the reduced affinity of proinsulin for the insulin recep- tors. The possibility of proinsulin conversion at the in- sulin receptor sites was investigated. Material recovered from the membranes upon dissociation of the proinsulin- receptor complex was intact proinsulin and did not ex- hibit any conversion by a variety of analytical methods. These results indicate that the lower affinity of pro- insulin for the insulin receptor in the liver is an intrinsic property of the proinsulin molecule. The lower uptake of proinsulin by the insulin receptor represents, in ad- dition to a slower degradation of the prohormone, a further mechanism by which proinsulin exerts prolonged, albeit reduced, action in vivo.
66 citations
TL;DR: A difference in the metabolic rate of the 2 hormones together with previously described differences in their hepatic extraction, are sufficient to explain the higher proinsulin :insulin ratio in serum compared to the pancreas.
Abstract: The disappearance of endogenous serum proinsulin and insulin was measured in 3 patients following removal of beta-cell adenomas of the pancreas. The mean half-disappearance time for proinsulin (17.2 min) was much longer than for insulin (4.8 min). This difference in the metabolic rate of the 2 hormones together with previously described differences in their hepatic extraction, are sufficient to explain the higher proinsulin :insulin ratio in serum compared to the pancreas.
60 citations
TL;DR: Evidence that glucose regulates total islet protein synthesis at the level of initiation includes increased [3H]leucine incorporation into nascent peptide with the distribution on heavier polysome aggregates indicating an increased number of ribosomes per mRNA, even when new RNA synthesis is blocked.
TL;DR: Active B-cell secretion composed of proinsulin, C-peptide and presumably insulin is present during insulin therapy in gestational, in some juvenile-onset-diabetic, pregnant women and in their offspring.
Abstract: Proinsulin is converted to insulin and C-peptide in the pancreatic islet cells; the latter two polypeptides are then secreted in equimolar concentration. Thus, measurement of C-peptide may provide an alternative means of studying B-cell function in pregnant, insulin-treated, diabetic patients and in their newborn infants (IDM). Using this approach, B-cell function was studied in eight normal, ten juvenile onset and five gestational diabetic patients at the time of delivery. Normal maternal and cord CPR (C-peptide immunoreactivity levels, mean ± SEM, were 1.5 ± 0.3 and 0.9 ± 0.1 ng/ml, respectively. In six of ten juvenile onset diabetics, CPR levels were undetectable, while in four CPR was elevated. Five gestational diabetic women had elevated CPR levels. Cord CPR levels were significantly higher in all IDM than in the normal controls. A prompt rise in CPR was seen following intravenous glucose load in four IDM. Gel filtration of selected sera demonstrated both C-peptide and proinsulin, the latter protein being bound to circulating insulin antibodies which have crossed the placenta. Thus, active B-cell secretion composed of proinsulin, C-peptide and presumably insulin is present during insulin therapy in gestational, in some juvenile-onset-diabetic, pregnant women and in their offspring.
TL;DR: Crystals appropriate for detailed X-ray crystal structure analysis of proinsulin using the known stereochemistry of insulin using the use of rigid-body vector search methods are grown.
Abstract: SINCE the first crystallisation of proinsulin in this laboratory1, we have tried to grow crystals appropriate for detailed X-ray crystal structure analysis. None of the pro-insulin crystal forms prepared proved ideal2, and we attempted to cocrystallise insulin and proinsulin. Such crystals, if both components were in ordered array, could provide a basis for the determination of the structure of proinsulin by the use of rigid-body vector search methods3,4 using the known stereochemistry of insulin5.
TL;DR: It would seem, therefore, that the genetic lesion responsible for the NZO state lies within the islets of Langerhans but not within the beta-cell, as yet unknown.
Abstract: New Zealand Obese (NZO) mice are characterised by hyperglycaemia, hyperinsulinaemia and obesity. The blood glucose and plasma insulin levels of these mice can be lowered to levels approaching those normally associated with non-obese mice, by the intra-peritoneal implantation of pancreatic islets from albino mice which have had theirβ-cells selectively destroyed by streptozotocin. Removal of these implanted islets causes the animals to revert to their original state. It would seem, therefore, that the genetic lesion responsible for the NZO state lies within the islets of Langerhans but not within the beta-cell. Its exact location is as yet unknown.
TL;DR: It was shown that high purification of porcine NPH insulin is of importance in reducing insulin dose, lipoatrophy and insulin binding capacity of serum.
Abstract: Side effects of insulin treatment (red spots at the injection site, lipoatrophy and insulin antibody formation) are primary related to the purity and the species of the insulin preparations used in treatment. To evaluate the clinical significance of these factors daily insulin requirements were registered in 1685 protamine insulin treated cases; red spots and lipoatrophy recorded in 500 NPH insulin treated patients and insulin antibodies determined in 173 patients. 51 of the patients were treated for more than 12 months with highly purified porcine insulin (proinsulin contamination <0.5 mmol/mol). 96 patients were treated for 1–4 years with porcine NPH insulin (proinsulin contamination 12–13 mmol/mol), and most of the remaining patients have been treated with mixed porcine-bovine insulin preparations in earlier periods. It was shown that high purification of porcine NPH insulin is of importance in reducing insulin dose, lipoatrophy and insulin binding capacity of serum. Thus insulin antibodies could be demonstrated in only 3 of 51 patients after 11/2 year of treatment with highly purified porcine NPH insulin. None of these patients developed red spots at the injection site and only one patient developed a small shallow depression of the subcutaneous tissue on one thigh. The daily insulin requirement was significantly lower when compared with the insulin requirement of a porcine NPH insulin treated control group.
TL;DR: Synthetic polypeptides of human C-peptide, [64-formyllysine]-human proinsulin 31–65 (II) and [41-glutamic acid, 43- glutamine, 64- formyllysines]- human proins insulin 31-65 (III) are described.
TL;DR: The glucose-like enhancing action of theophylline on both 45Ca2+ uptake and proinsulin synthesis could be due, in part at least, to a cyclic AMP-mediated stimulation of glycogenolysis.
TL;DR: Gel filtration appears at this point in time to be the most reliable method for assay of plasma proinsulin-like material, although the enzyme assay has significant limitations both in screening of normal subjects as well as in diagnosing insulinomas.
Abstract: Our laboratory has had a good experience using the gel filtration assay for proinsulin. Since the proteolytic enzymatic assay for proinsulin has the reported advantage of being simpler and requiring less plasma per assay, a comparison was made of the enzyme and gel filtration assays for plasma proinsulin. The proteolytic enzyme was prepared from rat muscle and a 30 to 60 per cent ammonium sulfate fraction was used. The kinetics of the enzyme were characterized with regard to substrate concentration (proinsulin and insulin), enzyme concentration, time of incubation and effect of plasma. The studies of enzyme using labeled substrates revealed identical behavior as with cold substrates. The Km for insulin was .476 × 10 −6 M and for proinsulin was .956 × 10 −6 M. Lineweaver-Burk plot showed competitive inhibition between insulin and proinsulin. A noncompetitive inhibitor of the enzyme was identified in a plasma sample from a “normal” human subject. The plasma proinsulin assay was performed as described by Kitabchi et al. with the modification that each plasma had an internal control incubated with labeled insulin or proinsulin. It was seen that the activity of the enzyme was somewhat variable in different plasmas. In noninsulinoma samples of plasma, the per cent proinsulin varied between 0 and 72 per cent. In the insulinoma plasmas, the values were 30 to 143 per cent. Twenty-one samples were compared with the gel filtration assay for proinsulin. This latter procedure utilizes G-50 Sephadex columns with size separation of proinsulin and insulin. Enzymatic assay values appeared consistently higher but still had a degree of correlation with those obtained by column. However, the enzyme assay has significant limitations both in screening of normal subjects as well as in diagnosing insulinomas, since false high proinsulin values may be found. Gel filtration appears at this point in time to be the most reliable method for assay of plasma proinsulin-like material.
TL;DR: Insulin synthesis, found to be stimulated by glucose like insulin secretion, was unaffected by all 3 gastrointestinal hormones whether or not glucose was present; and pancreozymin was the only substance to stimulate insulin secretion.
Abstract: Secretion and biosynthesis of (pro-) insulin were studied in isolated pancreatic mouse islets in the presence of secretin, pancreozymincholecystokinin, gastrin and glucagon, at 0, 100 and 300 mg⁄100 ml glucose. Incorporation of aHleucine into the proinsulin and insulin fraction obtained after gel nitration of the islet proteins on a Sephadex G 50 fine column was taken as parameter of insulin synthesis. Specific radioactivity of free intracellular leucine pool in the isolated islets was determined under different conditions. In the absence of glucose, pancreozymin was the only substance to stimulate insulin secretion.When glucose was present in the medium, glucagon was able to potentiate insulin output. Secretin and gastrin did not change insulin release. Insulin synthesis, found to be stimulated by glucose like insulin secretion, was unaffected by all 3 gastrointestinal hormones whether or not glucose was present. (Pancreatic) glucagon, on the other hand, potentiated glucose-induced insulin synthesis. Spe...
TL;DR: Single component porcine [ 125I] insulin and bovine [125I]proinsulin injected in rats were converted into high molecular weights forms that were reactive with guinea pig antisera against crystalline insulin although their immunoreactivity was diminished.
Abstract: Single component porcine [125I] insulin and bovine [125I]proinsulin injected in rats were converted into high molecular weights forms. These high molecular weight forms were reactive with guinea pig antisera against crystalline insulin although their immunoreactivity was diminished (65 to 75%) compared to that of control [125I]insulin and [125I]proinsulin (over 95%). Within 2 to 4 min of injection about 20% of the total immunoprecipitable radioactivity of rat sera appeared in the high molecular weight region (70,000 to 100,000 daltons) and within 2 hr it represented over 80%. A 4-hr incubation of control [125I]insulin and [125I]proinsu-lin with rat sera in vitro did not result in the production of high molecular weight forms. Denaturation with 8M urea-2% SDS did not affect significantly the high molecular weight forms of insulin or proinsulin. When denaturation was combined with sulfitolysis about 95% of the radioactivity of the high molecular weight [125I]insulin appeared in the A- and B-chain regions, s...
TL;DR: Results indicate that the insulin-degrading activity present in the islet tumor is, in fact, GIT and that the protease(s) that further catabolizes the insulin A and B chains is apparently missing in the insulinoma.
Journal Article•
TL;DR: Serum samples from a patient with a presumed islet-cell adenoma with a 20-year history of hypoglycaemic attacks were chromatographed on a 'Sephadex G50' column and a third immunoassayable and bioassaysable peak was present in serum taken fasting and after oral glucose.
TL;DR: The identity of the newly synthesized protein was proved by its high specific radioactivity, a molecular weight of 10 000, co-chromatography with porcine insulin after tryptic digestion, and the failure of cleavage of the original, non-trypsinated protein into two chains.
TL;DR: The insulin binding sites demonstrated by the immunoferritin technique resembled the insulin receptor of fat cell membranes in so far as they showed trypsin sensitivity, heat lability and temperature dependency of insulin dissociation.
Abstract: The localization of insulin on the surface of fat cell ghosts, incubated at physiological concentrations of the hormone, was tested with ferritin labelled insulin antibodies. Insulin binding sites, as revealed by this approach, were found to be uniformly distributed around ghost membranes. About 20% of the number of vesicles or 15% of the membrane surface area of a ghost preparation was tagged with ferritin. No ferritin labelling was observed when insulin was omitted from the reaction mixture or when it was replaced by proinsulin, insulin A and B chain, glucagon, ACTH or oxytocin. The membranes were also devoid of ferritin when the antiinsulin conjugate was substituted by unspecificγ-globulin conjugate. The insulin binding sites demonstrated by the immunoferritin technique resembled the insulin receptor of fat cell membranes in so far as they showed trypsin sensitivity, heat lability and temperature dependency of insulin dissociation. From the distance between the ferritin particles and the membrane surface it appears that the insulin binding sites are located very close to or within the ghost membrane. The number of ferritin particles on individual membranes per unit area was higher than the number of insulin receptors calculated from the data of insulin binding studies on the basis of equal distribution of the receptor sites.
TL;DR: 13 patients with classical hyperinsulinism of the islets of Langerhans had benign or malignant tumors or hyperplasia, confirmed by operation or biopsy, one of nine adults and one of four infants died.
Abstract: Classical hyperinsulinism is a relatively uncommon disease of the islets of Langerhans, characterized by hypersecretion of insulin and, sometimes, proinsulin. Neuropsychiatric symptoms and obesity predominate. All 13 of our patients had benign or malignant tumors or hyperplasia, confirmed by operation or biopsy. One of nine adults and one of four infants died. The latter, if they fail to respond to glucose, require emergency surgery. ( JAMA 230:1538-1543, 1974)
TL;DR: A method is described for extracting nanogram to microgram amounts of insulin and proinsulin by affinity binding to anti-insulin globulins coupled to agarose beads, readily applicable to other biologic fluids and extracts.