Showing papers on "Proinsulin published in 1978"

A bacterial clone synthesizing proinsulin.

Abstract: We have cloned double-stranded cDNA copies of a rat preproinsulin messenger RNA in Escherichia coli chi1776, using the unique Pst endonuclease site of plasmid pBR322 that lies in the region encoding amino acids 181-182 of penicillinase. This site was reconstructed by inserting the cDNA with an oligo(dG)-oligo(dC) joining procedure. One of the clones expresses a fused protein bearing both insulin and penicillinase antigenic determinants. The DNA sequence of this plasmid shows that the insulin region is read in phase; a stretch of six glycine residues connects the alanine at position 182 of penicillinase to the fourth amino acid, glutamine, of rat proinsulin.

Nonsuppressible insulin-like activity (NSILA) from human serum: recent accomplishments and their physiologic implications.

Abstract: It is now known that nonsuppressible insulin-like activity extracted from human serum (NSILA-S) consists of at least two chemically and biologically closely related polypeptides with a molecular weight of 7500. Their primary and tertiary structures strikingly resemble that of human proinsulin and suggest a common phylogenetic ancestor. Beyond their acute insulin-like actions on insulin target tissues the two polypeptides exert pronounced effects on growth of cultured cells and on sulfation, RNA and protein synthesis of cartillage. For these reasons they have been termed insulin—like growth factors (IGF I and II). In adipose tissue, their biologic potency is ∼60 times lower than that of insulin and corresponds to their weak affinity for the insulin receptor rather than to their high affinity for a specific IGF-receptor whose functional role is still unclear. By contrast, their biologic potency in heart and skeletal muscle is close to that of insulin ( 1 2 – 1 5 ) although their affinity for the insulin receptor is ∼100 times lower than that of insulin. Therefore, the insulin-like actions of IGFs on muscle appear to be mediated by the IGF-receptor. In fibroblasts and chondrocytes, binding of IGFs to specific high affinity IGF-receptors correlates closely with their effects on growth indices and sulfation. The latter effects occur at ∼50 times lower concentrations than with insulin. In native blood, IGFs are tightly associated with a specific carrier protein and the free forms are barely detectable. Binding to the carrier blocks their action on insulin target tissues, which explains the absence of acute insulin-like effects of endogenous IGF in vivo. In contrast, tissues concerned with growth may possess mechanisms to “extract” IGFs from the carrier complex. IGFs are under the control of growth hormone: total IGF levels are increased in acromegalics and decreased in hypopituitary and Laron dwarfs. This characteristic and their distinct biologic properties coin IGFs as members of the somatomedin family. The close relationship is further underlined by the pronounced cross-reactivity with somatomedins in radioreceptor and radioimmunoassays. Low serum IGF values are also found in patients with liver cirrhosis. Among other evidence, this points to the liver as one site of production of IGFs. Total IGF levels were not found to be elevated in 15 patients with hypoglycemia caused by extrahepatic tumors. Antibodies that allow to discriminate between the two species of IGF have recently been raised in rabbits. From the results of the radioimmunoassays, it becomes apparent that normal human serum contains 4–6 times more IGF II than IGF I. In acromegalic patients, only IGF I is elevated, whereas IGF II lies within the normal range. In hypopituitary and in Laron dwarfs, but also in patients with liver cirrhosis, IGF I is more drastically reduced than IGF II. Thus, IGF I may be the more important growth factor, and the two seem to be regulated differently. Besides carrier-bound IGFs, another large molecular weight peptide with nonsuppressible insulin-like activity is present in human serum. It is not interconvertible to small molecular weight IGF and it is not active on sulfation of cartilage. Therefore, it represents an entity different from IGFs. Thus, bioassays carried out in whole serum detect different insulin-like activities: Since carrier-bound IGF is inactive on adipose tissue the fat pad assay measures large molecular weight NSILA rather than IGF, whereas the sulfation assay reflects the serum level of carrier-bound IGFs and other somatomedins.

Mapping of the residues responsible for the negative cooperativity of the receptor-binding region of insulin

Abstract: Insulin binding to its receptor leads to negatively cooperative interactions among the receptor sites. Studies with 29 insulin analogues (animal insulins and proinsulin, insulin-like growth factor and chemically modified insulins) which vary 1,000-fold in their affinity for the receptor and in their biological potency, suggest that a discrete invariable region on the surface of the insulin monomer is responsible for inducing the negative cooperativity. This domain comprises some of the eight carboxy-terminal residues of the B-chain and the A21 asparagine. Burying of this ‘cooperative site’ in the dimerisation of insulin leads to a loss of negative cooperativity. A revised mapping of the insulin molecule is proposed, featuring distinct bioactive and cooperative sites.

Detection and kinetic behavior of preproinsulin in pancreatic islets

Abstract: Newly synthesized rat islet proteins have been analyzed by polyacrylamide slab gel electrophoresis and fluorography. A minor component having an apparent molecular weight of 11,100 was identified as preproinsulin by the sensitivity of its synthesis to glucose, the pattern of NH2-terminal leucine residues, and the rapidity of its appearance and disappearance during incubation of islets or islet cell tumors. A small amount of labeled peptide material which may represent the excised NH2-terminal extension of preproinsulin or its fragment was also detected. The kinetics of formation and processing of the preproinsulin fraction were complex, consisting of a rapidly turning over component having a half-life of about 1 min and a slower minor fraction that may have bypassed the normal cleavage process. The electrophoretic resolution of the preproinsulin and proinsulin fractions into two bands each is consistent with the presence of two closely related gene products in rat islets rather than intermediate stages in the processing of these peptides.

Insulin receptors in human endothelial cells: identification and characterization.

Abstract: Specific binding of 125I-insulin was found in cultured human endothelial cells obtained from human umbilical veins. The binding reaction was rapid and reversible, demonstrated receptor site-site interactions of the negatively cooperative type, and was dependent on the temperature, pH and duration of incubation. At 21 degrees C, steady-state conditions of binding occurred in 90 minutes, the pH optimum was 7.8 and less than 10% of the labeled hormone was degraded. Binding of tracer amounts of 125I-insulin was inhibited by concentrations of unlabeled insulin as low as 0.2 ng/ml and 50% inhibition was obtained at 2-5 ng/ml of unlabeled insulin. Unlabeled porcine insulin, porcine proinsulin and desoctapeptide insulin inhibited the binding of 125I-porcine insulin in direct proportion to their biological potencies, whereas to the cells was inhibited by antibodies against insulin receptors. We conclude that human endothelial cells possess specific receptors for insulin whose physio-chemical properties are similar to those of insulin receptors in other tissues.

Insulin resistance, acanthosis nigricans, and normal insulin receptors in a young woman: evidence for a postreceptor defect.

Abstract: We have previously described a group of young females with virilization, acanthosis nigricans, insulin resistance, and markedly decreased binding of insulin to its receptor (syndrome of insulin resistance and acanthosis nigricans type A). The present report concerns a 15-yr-old female with clinical features indistinguishable from the type A patients, including virilization, acanthosis nigricans, and extreme resistance to endogenous and exogenous insulin. Insulin levels were 400-650 microU/ml while fasting and were over 2200 microU/ml when stimulated. Proinsulin was less than 10% of the total immunoassayable insulin. In distinct contrast to the type A patients, insulin receptors on cells from this patient were entirely normal on the basis of specificity, negative cooperativity, affinity, concentration, and interaction with antiinsulin receptor antibodies. These findings suggest the presence of an intracellular defect as the cause of the observed insulin resistance.

On the role of the proinsulin C-peptide.

Abstract: Intracellular cleavage of protein and polypeptide precursors is now recognized as a widely occurring biosynthetk: mechanism. As this field has developed, proinsulin and its cleavage patterns and secretory products have served as useful models for investigations of other systems. A particularly relevant aspect of the proprotein concept is the simple mechanism it provides for the coordinate synthesis and discharge of related peptides from endocrine or other secretory cells. This report reviews briefly the role of the proinsulin C-peptide, first in terms of its special biosynthetk: functions, which are unique to the assembly of the two-chain insulin structure, and then with regard to its more general implications for other biosynthetk and secretory systems.

The effect of sugars on (pro)insulin biosynthesis

Abstract: Rates of incorporation of [4,5-(3)H]leucine into insulin plus proinsulin, designated ;(pro)insulin', and total protein in rat pancreatic islets were measured. Glucose stimulates rates of total protein and (pro)insulin biosynthesis, but (pro)insulin biosynthesis is stimulated preferentially. Mannose and N-acetylglucosamine also stimulate (pro)insulin and total protein biosynthesis; inosine and dihydroxyacetone stimulate (pro)insulin biosynthesis specifically. Fructose does not stimulate (pro)insulin biosynthesis when tested alone, but does so in the presence of low concentrations of glucose, mannose or N-acetylglucosamine. Many glucose analogues do not stimulate (pro)insulin biosynthesis. Mannoheptulose inhibits synthesis of (pro)insulin and total protein stimulated by glucose or mannose but not by dihydroxyacetone, inosine or N-acetylglucosamine; phloretin (9mum) inhibits N-acetylglucosamine-stimulated (pro)insulin biosynthesis preferentially. The data are in agreement with the view that the same glucose-sensor mechanism may control both insulin release and biosynthesis, and ;substrate-site' model is suggested. The threshold for stimulation of biosynthesis of (pro)insulin and total protein is lower than that found for glucose-stimulated insulin release; moreover the biosynthetic response to an elevation of glucose concentration is slower than that found for insulin release. The physiological implication of these findings is discussed. Caffeine and isobutylmethylxanthine, at concentrations known to increase islet 3':5'-cyclic AMP and potentiate glucose-induced insulin release, were without effect on rates of glucose-stimulated (pro)insulin biosynthesis.

The insulin receptor as a universal marker of activated lymphocytes.

Abstract: Although resting, rat thymus-derived spleen lymphocytes do not bear insulin receptors, these receptors emerge upon the membrane of alloimmune rat T cells subsequent to skin grafting. These studies examine conditions that permit the emergence of lymphocyte insulin receptors upon T or B-enriched rat lymphocyte populations. Allogeneic stimulation was accomplished in vivo by skin grafting from (Lewis × Brown Norway)F1 (LBN) to Lewis male rats or by Graft-vs.-host (GVH) reaction established by intraperitoneal injections of 2 × 108 Lewis leukocytes each week for a total of four injections into LBN animals. In vitro allogeneic stimulation was provided by one-way mixed lymphocyte cultures between the same strains. Lastly, both T and B-enriched populations were interacted with mitogens concanavalin A (Con A), phytohemagglutinin (PHA-P), and lipopolysaccharide (LPS). The T-enriched populations were highly enriched for T lymphocytes (90%) and macrophage-depleted while B-enriched populations contained nearly 85% B cells. Insulin receptors emerged upon T-enriched cell populations consequent to allogeneic skin grafting with saturability, specificity and high affinity (kd = 1.1 nM) as well as after GVH. Responder strain lymphocytes developed an insulin receptor during allogeneic mixed lymphocyte cultures within 72 h of initiation. Specific insulin-binding sites also appeared upon T-enriched populations after culture with PHA-P and Con A but not LPS. Conversely, B cells developed an insulin receptor after interactions with LPS but not PHA-P or Con A. Anti-Ig and complement but not rat anti-T cell antibody abrogated the ability of LPS to induce an insulin receptor on B-enriched cells. Binding specificity was demonstrated by the inhibition of [125I]-labeled insulin in the order porcine insulin ≊ desalanine insulin > proinsulin ≫ desoctapeptide insulin with growth hormone exhibiting no such inhibition. These data demonstrate that a true insulin receptor appears upon both T and B cells consequent to activation. The lymphocyte insulin receptor is a universal marker of cellular activation as it is not restricted to clone or species and may be applied to B and T lymphocytes of mouse, rat and man.

Characterization of proinsulin-insulin intermediates in human plasma.

Abstract: This work addressed the problem of heterogeneity of immunoreactive insulin (IRI) in human plasma. Subjects with normal glucose tolerance were given 75g of an oral glucose solution, followed in 30 min by an intravenous infusion of 30g of arginine over 30 min. At the end of the infusion blood was withdrawn for analysis. IRI was extracted from plasma of individual subject by immunosorbent columns and was fractionated by gel filtration, disc gel electrophoresis and isoelectric focusing. Human IRI components were identified by molecular size, immunoreactivity with a human proinsulin antibody, sensitivity to trypsin, and by comparison of electrophoretic mobility and isoelectric point with porcine pancreatic products, after suitable correction for electric charge and molecular weight differences. The pattern of IRI heterogeneity was the same among six healthy subjects. Heterogeneity of proinsulin-size IRI in circulation was more marked than that of insulin-size material. Proinsulin and desdipeptide proinsulin were present in approximately equal amounts accompanied by minor amounts of split proinsulin and monodesamido-desdipeptide proinsulin. Insulin-size IRI contained over 80% insulin. Minor amounts of monodesamidoinsulin and diarginylinsulin were observed in some cases. The types of IRI components observed in plasma are evidence in support of a physiologic role of trypsin-and carboxypeptidase B-like enzymes in the conversion of proinsulin to insulin. Moreover, this study provides a base line for investigation of abnormalities in proinsulin-to-insulin conversion that may be associated with certain pathologic states.

Kinetics of human C-peptide in man.

Abstract: A review of studies concerned with the metabolism of human C-peptide showed that the kidney is the major organ in C-peptide removal. The turnover rate of C-peptide was similar to that of proinsulin but considerably slower than that of insulin. An analysis of corresponding C-peptide and insulin concentrations in normal subjects after the administration of oral glucose or intravenous glucagon was used to define the relationships between the two peptides. These results show that different peripheral vein concentrations of insulin may correspond to identical C-peptide concentrations, depending on sampling conditions.

beta-cell function in children with diabetes.

Abstract: Although it is generally believed that insulin secretion is minimal or absent in juvenile-onset diabetes, we have found appreciable levels of C-peptide at the time of onset in 12 patients, 4 to 16 years old (9.3 ± 4.2). Ten of them had ketonuria but none severe ketoacidosis. All entered a remission period. Most of the patients had near normal C-peptide levels during the remission, and their beta cells had the capacity to respond to a breakfast stimulation with increased insulin secretion. C-peptide and proinsulin were also determined in 98 juvenile diabetics with age at onset of 1 to 16 years (6.8 ± 3.9) and a duration of diabetes between two and 17 years (6.7 ± 3.4). Many were found to have persisting beta-cell function, which seems to be of importance for ensuring stability in metabolic control. Although little is known about factors that may slow or reverse the process leading to beta-cell failure, our results suggest that early detection and intensive treatment of diabetes before severe metabolic disturbances have occurred may help preserve beta-cell function.

Insulin binding to solubilized material from fat cell membranes: Evidence for two binding species

Abstract: The components of fat cell membranes responsible for the binding of insulin were solubilized by treatment with the nonionic detergent Triton X-100. By using a polyethylene glycol precipitation method to assay specific insulin binding, the soluble preparation was shown to have insulin-binding characteristics similar to those of intact fat cells. Further studies of this preparation by polyacrylamide gel electrophoresis in the presence of 125I-labeled insulin demonstrated two distinct insulin binding activities, designated species I and II. The two species were separated by electrophoresis in the absence of iodo-labeled hormone and eluted from the gel. Scatchard analysis of the insulin binding data for species I showed a curvilinear plot with the initial portion having a Kd of 1.3 × 10-10 M. The Scatchard plot for species II was linear with a Kd of 6.0 × 10-9 M. Desoctapeptide insulin and glucagon failed to compete for the insulin-binding sites in both species whereas desalanine insulin was an effective competitor. High concentrations of proinsulin competed with the iodo-labeled hormone for binding to species I but not to species II. In the presence of a low concentration of 125I-labeled insulin (0.3 nM) some species I activity appeared to be converted to species II activity; there was no evidence of interconversion between the two species in the absence of insulin. Neither species degraded insulin as measured by trichloroacetic acid precipitation or rebinding to intact fat cells. These findings indicate the existence in the adipocyte plasma membrane of two insulin-binding species that have distinct physicochemical properties.

C-peptide and Insulin in Liver Disease

Abstract: The liver is the primary organ of insulin clearance; thus, hyperinsulinism in peripheral blood may result from diminished insulin degradation rather than hypersecution. Proinsulin is cleaved in the pancreatic β-cell to insulin and C-peptide, which are released into the circulation in equimolar quantities. Unlike insulin, C-peptide is not degraded by liver, and in liver disease the peripheral C-peptide concentration is a better index of insulin secretion than is peripheral insulin concentration. Fasting plasma insulin concentration was raised in 21 cirrhotic patients (0.13 ± 0.02 vs. 0.06 ± 0.01 nmol per liter) despite normal fasting blood glucose concentrations. C-peptide concentrations and, therefore, insulin secretion did not differ from controls9, and the C-peptide:insulin ratio was decreased in cirrhotic subjects (4.0 ± 0.5 vs 7.0 ± 1.1). After oral glucose, hyperinsulinemic cirrhotic subjects had similar C-peptide concentrations to those of cirrhotics with a normal insulin response in whom glucose values were similar. Hyperinsulinism (both fasting and after glucose) appears to result from diminished insulin degradation, therefore. To distinguish the roles of liver damage and of portal-systemic shunting, similar studies were performed in patients with ostensibly normal liver function but long-standing portal venous block. Despite mild hyperglycemia, hyperinsulinism was not found in these subjects, either fasting or after oral glucose. The fasting C-peptide:insulin ratio was normal, and only after stimulation of insulin secretion by glucose was a mild impairment of insulin degradation seen. The hyperinsulinism of hepatic cirrhosis is due to impaired insulin degradation; this is more likely to result from liver damage per se than from portal-systemic shunting.

The stimulus-secretion coupling of glucose-induced insulin release. Metabolic and functional effects of NH4+ in rat islets.

Abstract: NH4+ caused a dose-related, rapid, and reversible inhibition of glucose-stimulated insulin release by isolated rat islets. It also inhibited glyceraldehyde-, Ba2+-, and sulfonylurea-stimulated insulun secretion. NH4+ failed to affect glucose utilization and oxidation, glucose-stimulated proinsulin biosynthesis, the concentration of ATP, AD, and AMP, and the intracellular pH. NH4+ also failed to affect the ability of theophylline and cytochalasin B to augment glucose-induced insulin release. However, in the presence and absence of glucose, accumulation of NH4+ in islet cells was associated with a fall in the concentration of NADH and HADPH and a concomitant alteration of 86Rb+ and 45Ca2+ (or 133Ba2+) handling. These findings suggest that reduced pyridine nucleotides, generated by the metabolism of endogenous of exogenous nutrients, may modulate ionophoretic processes in the islet cells and by doing so, affect the net uptake of Ca2+ and subsequent release of insulin.

Role of zinc and calcium in the formation and storage of insulin in the pancreatic β-cell

Abstract: 1. The effects of culture of isolated mouse islets of Langerhans for up to 9 days in media which had been depleted of zinc electrochemically or with the chelating agent Tris-(2-aminoethyl) amine, or of calcium, have been compared. 2. An 83% reduction of extracellular zinc concentration did not adversely affect proinsulin biosynthesis, conversion of proinsulin to insulin, or the ability of cells to store newly formed insulin in granules. When incubation media were depleted of both zinc and calcium the β-cells produced abnormally large electron-lucent granules, consistent with the failure of insulin to crystallise within the granule sac. 3. Very similar results, with formation of large electron lucent granules, were obtained after culture of islets in the absence of calcium but in the presence of normal concentrations of zinc. 4. It is suggested that zinc may play a less critical role in the biosynthesis of proinsulin and its conversion to insulin, while calcium may have a more important function in insulin storage, than has sometimes previously been supposed.

Synthesis of Carp Proinsulin in Xenopus Ocytes

Abstract: Total poly(A)-containing RNA from Brockmann bodies of carp (Cyprinus carpio) directs the synthesis of authentic carp proinsulin in Xenopus oocytes Neither preproinsulin nor further processing of pronisulin to insulin can be detected in the oocytes

Insulin, C-peptide, and Proinsulin in Nondiabetics and Insulin-treated Diabetics: Characterization of the Proinsulin in Insulin-treated Diabetics

Abstract: Immunoreactive insulin (IRI), C-peptide, and proinsulin were determined in 46 fasting nondiabetic persons. The means ± S.D. for IRI, C-peptide, and proinsulin (in pmol/ml.) in the 46 nondiabetics were 0.054 ± 0.027, 0.39 ± 0.12, and 0.014 ± 0.009, respectively. The molar ratio between C-peptide and IRI varied from 4.0 to 16.7, with a mean value of 7.9 ± 3.4. Proinsulin constituted 5–36 per cent of IRI, with a mean of 20 ± 10 per cent. Sera from 24 diabetics aged 17–67 years treated with conventional crystalline insulin for durations of from one to 29 years (mean: 15 years), all of whom had circulating insulin antibodies, were extracted with acid ethanol. Total IRI varied from 2.0 to 22.6 pmol/ml. C-peptide was detectable in only three patients (0.05, 0.08, and 0.15 pmol/ml.). In contrast, human proinsulin was found in all patients and ranged from 0.09 to 9.4 pmol/ml., corresponding to 1 to 45 per cent of total IRI. Species-specific immunoassays for bovine and porcine proinsulin revealed the presence of bovine proinsulin in 23 of the patients (mean 0.36 ± 0.20 pmol/ml.) and porcine proinsulin in eight patients (mean 0.15 ± 0.13 pmol/ml.). Total proinsulin constituted 5 to 64 per cent, mean 22 per cent, of total IRI, while human proinsulin amounted to 1 to 45 per cent, mean 10 per cent, of IRI. Although no insulin secretion could be detected in 21 of the patients, as judged by the amount of C-peptide, the B-cell was still able to secrete proinsulin. Insulin antibodies bind a portion of the secreted proinsulin, thus prolonging its half-life and increasing its concentration in serum.

Multiplication-stimulating activity (MSA): a somatomedin-like polypeptide from cultured rat liver cells.

Abstract: Multiplication-stimulating activity (MSA) has been purified to homogeneity with the use of Dowex chromatography. Sephadex gel filtration, and preparative disc gel electrophoresis. A molecular weight of 8,700 was determined for both native and reduced and alkylated MSA by chromatography on a 6% agarose column in 6 M guanidine HCl. Amino acid analysis of performic acid-oxidized MSA revealed 2--3 cysteic acid residues, and reduction and alkylation resulted in loss of biologic residues, and reduction and alkylation resulted in loss of biologic activity. These results suggested the presence on one intrachain disulfide bond, which is required for biologic activity. Specific receptors for MSA have been identified on chick embryo fibroblasts, human skin fibroblasts, a rat liver cell line, and purified rat liver plasma membranes. The closely related peptides, acid ethanol-soluble nonsuppressible insulin-like activity (NSILA-S) and somatomedin-A, competed for MSA tracer binding to these MSA receptors, whereas unrelated peptides did not compete. MSA receptors were divided into two types based on their reactivity with insulin and proinsulin. Insulin and proinsulin competed potently for MSA binding to chick embryo and human skin fibroblasts (type II) but did not compete for MSA tracer binding to rat liver cells and purified rat liver membranes (type I). In chick embryo fibroblasts, the concentrations of MSA, insulin, proinsulin, and somatomedin-A, which inhibited [125I]MSA binding by 50%, also gave half-maximal stimulation of DNA synthesis, consistent with the MSA receptor being a mediator of DNA synthesis. MSA tracer has also been shown to bind specifically to rat serum proteins. The MSA binding profile on Sephadex G-200 was shown to be growth hormone dependent. Since the serum half-life of somatomedin activity in the rat was also growth hormone dependent, these results suggest that growth hormone induces the binding protein for somatomedin and thereby governs the half-life of somatomedin. Finally, the ability of two rat liver cells lines to multiply in serum-free medium did not depend upon the level of MSA in the conditioned medium.

The handling of insulin, proinsulin, and C-peptide by the isolated rat kidney.

Abstract: The renal handling of proinuslin, insulin, and C-peptide was studied in the isolated perfused rat kidney. At perfusate peptide concentrations below 12 ng./ml. (control value), the organ clearance of proinsulin (637 ± 74 μl. per minute) was significantly (P When kidneys were perfused with insulin in concentrations above 330 ng./ml., the organ clearance fell to approximately half the control value and its fractional urinary excretion increased several fold, indicating saturation of luminal absorption of filtered insulin. As the fall in organ clearance of insulin was larger than any decrease in the rate of insulin filtration that may have occurred, we conclude that at high insulin concentrations saturation of contraluminal uptake of insulin also occurs. Similarly, the organ clearance of C-peptide declined to 46 per cent of the control level at perfusate concentrations of 108 to 301 ng./ml. In contrast, despite perfusing kidneys with concentrations of proinsulin as high as 367 ng./ml., its organ clearance and fractional urinary excretion did not decline. There was no evidence of conversion of proinsulin to insulin or C-peptide.

Absorption, effectiveness and side effects of highly purified porcine NPH-insulin preparations (Leo).

Abstract: The clinical characteristics of highly purified porcine NPH-insulin (Insulin Retard RI®) were investigated, including absorption from the subcutaneous tissue, blood glucose-lowering effect, stability of mixtures of NPH and regular insulin and measurement of circulating porcine proinsulin and insulin antibodies in diabetics. The absorption of NPH-insulin followed first order kinetics. The half time was 6.9±2.6 h, with an intraindividual coefficient of variation of 26% and an interindividual coefficient of variation of 55%. After 24 h 90% of the injected insulin had disappeared from the subcutaneous tissue. The plasma insulin concentration was maximal 4–5 h after the injection and 24 h after the injection it was not significantly higher than before the injection. The blood glucose-lowering effect was significant 2.5 h after subcutaneous injection of NPH-insulin and was maximal after 5.5 h. The blood glucose-lowering effect of a pre-prepared mixture of 70% NPH and 30% regular insulin was not significantly different from the effect of 70% NPH and 30% regular insulin injected separately, which indicates the stability of mixtures of NPH and Regular insulin. Porcine proinsulin disappeared from the serum of patients switched to treatment with highly purified porcine NPH insulin and the insulin antibody titer fell. It was concluded that insulin Retard RI® has a well-defined, reproducible effect with a clinically useful time course.

Insulinoma resection facilitated by the artificial endocrine pancreas.

Abstract: The artificial endocrine pancreas was adapted to assist with the intraoperative localization of an insulin-secreting islet cell adenoma in a 23-year-old patient with a five-month history of hypoglycemic attacks. Glycemia was monitored in continuously withdrawn whole blood, and dextrose was infused to maintain euglycemia by an artificial endocrine pancreas with a closed loop that excluded its usual insulin delivery capability. The dextrose infusion rate was established in accordance with a control algorithm whose parameters were chosen to amplify changes in dextrose delivery rate for small alterations in the measured baseline glucose concentration. The dextrose infusion rate preoperatively was 155 mg. per minute and decreased to 100 mg. per minute with initiation of surgery. An area in the tail of the pancreas suspected of containing the insulinoma was excluded from the circulation by a noncrushing clamp. After 14 minutes the dextrose infusion progressively decreased to 27 mg. per minute reflecting a glycemic rise of 15 mg. per deciliter. These changes were taken to represent a fall in ambient insulin activity. This was subsequently confirmed directly by the demonstration of reduction in immunoreactive insulin and progressive increase in both plasma free fatty acid levels and postoperative glucose intolerance. Exclusion of the insulinoma from the circulation resulted also in a rapid decrease of circulating proinsulin concentration giving an estimated half-life of 25 minutes.

Ectopic insulin and occam's razor: reappraisal of the riddle of tumour hypoglycaemia

Abstract: The concept of ectopic insulin production is challenged on the basis of a review of 120 cases from the literature on extrapancreatic tumours associated with hypoglycaemia in which insulin or insulin-like activity were measured. No case met two or more of five criteria of ectopic hormone production. The evidence indicates that hypoglycaemia of extrapancreatic tumours cannot be attributed to insulin. In those rare cases in which plasma insulin was reported as high, pancreatic beta-cells could not be excluded as the source of insulin. Interestingly, many of these dubious cases had carcinoid histology. The review also points out a close association between some spindle-cell tumours and carcinoid tumours which may be relevant to discussion on the disputed origin of some "mesothelial" tumours. Nonsuppressible insulin-like activity (NSILA) consists of a number of factors mimicking insulin activity which compete with insulin or proinsulin for membrane receptors and may crossreact in bioassays, immunoassays, and receptor assays. The question of whether one or several of these substances may be responsible for extrapancreatic hypoglycaemia remains to be elucidated.

Physical Characteristics of Insulin Receptors on Renal Cell Membranes

Abstract: Certain physical characteristics of the insulin-kidney-membrane interaction in dog renal cortex have been defined. Two classes of binding sites were identified: the high-affinity-low-capacity site had a Ka of 1.3 × 109 M1 and a capacity of 43 fmols per milligram membrane protein. The lower-affinity site Ka was 3.7×107 M-1. Saturabili t y of receptor sites could not be demonstrated at any insulin concentration. At 21° C, binding of insulin was maximal after 60 minutes of incubation, with a t1/2of proinsulin>desoctapep-tide insulin). Arginine vasopressin and glucagon did not compete with native (labeled) insulin for binding sites. The kinetic properties of the kidney insulin-receptor sites are similar to those previously reported for liver and fat cell receptors. A comparison of specific binding of 125I-insulin to kidney and liver membranes in the presence of various cation concentrations showed no enhancement of binding in either tissue by NaCl as high as 2 M and KC1 up to 50 mM. In contrast, 25 mM CaCl2 caused an increase in specific binding from 3.8 to 7.7 fmols 125I-insulin per milligram protein in kidney and from 1.0 to 2.1 fmols per milligram protein in liver. MgCl2 (10 mM) increased binding of insulin from 3.5 to 7.3 fmols per milligram in kidney and from 1.0 to 2.1 fmols per milligram in liver. EDTA (2mM) had no intrinsic effect on binding but reversed (by 50 per cent) the Ca2+ -mediated enhancement of binding. Divalent cations appeared to influence both high and low affinity sites. 125I-insulin degradation occurred as a function of total insulin concentration in the kidney membrane system and reduced the amount of “bindable” free hormone. The process was not saturable below 10-6 M insulin. Degradation effects influenced calculated equilibrium constants to a minimal degree.