Showing papers on "Proinsulin published in 1985"
TL;DR: Findings provide direct evidence that coated secretory granules are the major, if not the only, cellular site of proinsulin to insulin conversion and suggest that the Golgi stack is not involved in conversion, and that intercisternal transport and coated granule formation are hitherto unrecognized energy-requiring steps that precede conversion.
280 citations
TL;DR: It is suggested that insulin at physiological levels appears to increase bone collagen synthesis by a direct effect on the osteoblast, whereas insulin at high concentrations has an additional action to increase the replication of collagen-synthesizing cells.
Abstract: We characterized the effect of insulin on collagen synthesis in 21-day-old fetal rat calvaria maintained in organ culture. All experiments were done in the presence of 100 mg/dl glucose and 3 mM phosphate, which were found to be optimal concentrations for insulin responsiveness. All concentrations of insulin tested (1 nM to 1 microM) increased the percentage of collagen being synthesized in the central bone, whereas only high concentrations of hormone (100 nM to 1 microM) increased the percentage of collagen being synthesized in periosteum. Insulin at 3 nM increased the labeling of type I collagen in the central bone, but did not alter the labeling of type I or III collagen in the periosteum. Proinsulin was approximately 10-100 times less effective than insulin in stimulating collagen synthesis, whereas porcine relaxin and C-peptide were ineffective. Insulin did not enhance the deposition of newly synthesized collagen in the bone by a mechanism that involved decreasing the degradation of collagen. To determine whether insulin enhanced collagen synthesis by increasing the replication of collagen-synthesizing cells, we tested the effect of insulin in the presence of hydroxyurea, a DNA synthesis inhibitor. Hydroxyurea at 1 mM had little effect on collagen synthesis in control cultures or those treated with 1 or 10 nM insulin. However, hydroxyurea blunted the stimulation of collagen synthesis that occurred at higher concentrations of insulin. These experiments suggest that insulin at physiological levels appears to increase bone collagen synthesis by a direct effect on the osteoblast, whereas insulin at high concentrations has an additional action to increase the replication of collagen-synthesizing cells.
188 citations
TL;DR: The results suggest that the stimulatory compounds such as quercetin and (-)epicatechin may, at least in part, exert their effects on insulin release via changes in Ca2+ metabolism.
Abstract: The effects of some flavonoids, a group of naturally occurring pigments one of which has been claimed to possess antidiabetic activities, on insulin release and 45Ca2+ handling have been studied in isolated rat islets of Langerhans. Insulin release was enhanced by approximately 44-70% when islets were exposed to either (-)epicatechin (0.8 mmol/l) or quercetin (0.01-0.1 mmol/l); others such as naringenin (0.1 mmol/l) and chrysin (0.08 mmol/l) inhibited hormone release by approximately 40-60%. These effects were observed only in the presence of 20 mmol glucose/l. Quercetin (0.01 mmol/l) and (-)epicatechin (0.8 mmol/l) both inhibited 45Ca2+ efflux in the presence and absence of extracellular Ca2+. In the presence of 20 mmol glucose/l both the short-term (5 min) and steady-state (30 min) uptake of 45Ca2+ were significantly increased by either quercetin or (-)epicatechin. These results suggest that the stimulatory compounds such as quercetin and (-)epicatechin may, at least in part, exert their effects on insulin release via changes in Ca2+ metabolism.
177 citations
TL;DR: It is concluded that specific high affinity insulin receptors possessing tyrosine kinase activity are widely distributed in normal human ovary, and IGF-I receptors in normal ovary are either absent or present at very low density.
Abstract: Insulin-resistant hyperinsulinemic states are now widely known to be associated with ovarian hyperandrogenism, and this is thought to be due to an action of insulin on the ovary. However, the identity of the receptor that is responsible for insulin action in these patients, whose insulin receptors on classical target tissues are severely impaired, is unclear. We now report the presence of insulin receptors in stromal and follicular compartments as well as in granulosa cells obtained from normal ovaries. After 15-h incubations at 4 C with [125I]insulin and tissue fragments, specific insulin binding was 6-19% and 7-13%/mg protein (n = 8) to stroma and theca, respectively. Granulosa cells obtained in the course of in vitro fertilization were separated from red cells on a Percoll gradient; specific insulin binding ranged from 9-15%/10(6) cells. Insulin binding was characterized by sensitive insulin competition (half-maximal, 10 ng/ml), appropriately shifted proinsulin competition (20 times to the right), and complete inhibition by specific anti-insulin receptor antibodies (B-2). An antibody to the insulin-like growth factor I (IGF-I) receptor (alpha IR-3) that inhibits IGF-I binding to IGF-I receptors in other cell systems had no effect on insulin binding. Further proof that this binding is to classic insulin receptors was obtained from measurement of insulin-stimulated receptor autophosphorylation. When insulin receptors from stroma were extracted with Triton X-100 and incubated with [gamma-32P]ATP and Mn, insulin increased the incorporation of 32P into the beta-subunit of the receptor 5-fold. In parallel studies with [125I-]IGF-I and specific blocking antibodies to its receptor, no detectable IGF-I binding to stroma or follicles was found. We conclude that specific high affinity insulin receptors possessing tyrosine kinase activity are widely distributed in normal human ovary. IGF-I receptors in normal ovary are either absent or present at very low density. Binding of insulin to its own receptor (as opposed to IGF-I receptors) appears to be the most likely first step in the stimulation of ovarian steroidogenesis by insulin in normal ovaries and possibly in insulin-resistant states as well.
176 citations
TL;DR: It is concluded that proinsulin is inefficiently processed in the vascular compartment, and subcutaneous administration of the precursor can result in the formation of conversion intermediates with the potential for contributing to biological activity.
Abstract: Since a complete map of insulin-related peptides in humans requires consideration of proinsulin, Arg32/Glu33-split proinsulin, Arg65/Gly66-split proinsulin, des-Arg31,Arg32-proinsulin, des-Lys64, Arg65-proinsulin, and insulin, we applied high performance liquid chromatography coupled with radioimmunoassay to investigate the formation of proinsulin conversion intermediates in vitro and in vivo. Kinetic analysis of proinsulin processing by a mixture of trypsin and carboxypeptidase B (to stimulate in vivo processes) revealed (a) a rapid decline in proinsulin concommitant with formation of conversion intermediates, (b) formation of des-Arg31, Arg32-proinsulin and des-Lys64,Arg65-proinsulin in the ratio 3.3:1 at steady state, and (c) complete conversion of the precursor to insulin during extended incubation. Studies on normal human pancreas identified a similar ratio of des-Arg31,Arg32-proinsulin to des-Lys64,Arg65-proinsulin (approximately 3:1), whereas two insulinomas contained sizable amounts of des-Arg31,Arg32-proinsulin, but barely detectable amounts of des-Lys64,Arg65-proinsulin. None of the tissues contained measurable quantities of Arg32/Glu33- or Arg65/Gly66-split proinsulin. Analysis of plasma from three diabetic subjects managed by the intravenous infusion of human proinsulin revealed less than 1% processing of the circulating precursor to conversion intermediates and no processing of the precursor to human insulin. Nevertheless, analysis of plasma from the same subjects managed by the subcutaneous infusion of proinsulin revealed 4-11% processing of the precursor to intermediates that had the properties of des-Arg31,Arg32-proinsulin and Arg65/Gly66-split proinsulin. We conclude that (a) processing of proinsulin to insulin in vivo as in vitro likely occurs by preferential cleavage at the Arg32-Glu33 peptide bond in proinsulin, (b) proinsulin is inefficiently processed in the vascular compartment, and (c) subcutaneous administration of the precursor can result in the formation of conversion intermediates with the potential for contributing to biological activity.
86 citations
TL;DR: It is suggested that immunoassayable insulin detected in extrapancreatic tissues of the adult rat is synthesized by the pancreas.
Abstract: The purpose of these studies was to determine whether insulin detected immunochemically in extrapancreatic tissues of the adult rat is synthesized in situ by quantitating mRNA in these tissues. A blot hybridization assay was utilized with cloned 32P-proinsulin cDNA. The lower limit of detection was estimated to be 3pg. Proinsulin mRNA concentration was found to be 1000–1500 μg in isolated pancreatic islets and was easily detected in total pancreatic RNA at 10–15 pg/ μg. Proinsulin mRNA was quantitated in rat insulinoma cells adapted to culture at levels 1∶50 those in normal islets. Samples of RNA (20–50 μg) enriched about 50-fold for mRNA sequences by repeated oligo-deoxythymidylate chromatography were assayed. No insulin mRNA was detected in 50 μg samples of RNA from brain or in 20 μg samples from subsections of brain or other extrapancreatic tissues. RNA samples were undegraded as assessed by ability to stimulate protein synthesis in a cell-free system. Proinsulin mRNA from pancreas was added to brain homogenates and recovered intact. Brain RNA samples with insulin mRNA levels 1:1000 that of pancreas would be predicted to have 50–75 pg proinsulin mRNA/50 μg sample assayed if present. Because none was found, brain must have a concentration <1:6,000 that of pancreas. These findings suggest that immunoassayable insulin detected in extrapancreatic tissues of the adult rat is synthesized by the pancreas.
62 citations
TL;DR: The similarity in the thermodynamic parameters of both insulin species at the different pH's suggests that there are minimal structural changes at the monomer-monomer contact site over this pH range, and may suggest that the formation of the insulin dimer is not driven by hydrophobic bonding but, rather, is driven by the formation between subunits of four hydrogen bonds in an apolar environment.
58 citations
TL;DR: Leukocyte DNA from one member of a proinsulinemic family was analyzed and a point mutation that changed guanine to adenine in the insulin gene was found, which implies that a substitution of histidine for arginine has occurred at amino acid position 65.
Abstract: Familial hyperproinsulinemia is characterized by the accumulation of proinsulin-like material (PLM) in the plasma of affected patients This disorder is inherited in an autosomal dominant fashion The accumulation of PLM is thought to be due to the impaired conversion of proinsulin to insulin Although PLM has been suggested to have an amino acid substitution, it has been impossible to locate and identify a substituted amino acid, due to the difficulty in isolating sufficient amounts of PLM from plasma samples Therefore, we analyzed leukocyte DNA from one member of a proinsulinemic family, and we found a point mutation that changed guanine to adenine in the insulin gene This transition implies that a substitution of histidine for arginine has occurred at amino acid position 65 Furthermore, it indicates that arginine at 65 is essential for the conversion of proinsulin to insulin Our results suggest a novel mechanism by which disease can be incurred: a heritable disorder can result from a posttranslational processing abnormality caused by a point mutation
56 citations
TL;DR: Since the direct assay sensitivity was not sufficient for measurement of endogenous hPI levels, a simple procedure for quantitative extraction of proinsulin-like material (PLM) from up to 40 ml of plasma on insulin antibody-Sepharose columns was developed, suitable for measuring endogenous PLM in extracted plasma samples.
Abstract: A radioimmunoassay for human proinsulin (hPI) has been developed using biosynthetic hPI prepared by recombinant DNA technology as immunogen, standard, and tracer. The antiserum was raised in a guinea pig and then adsorbed against insulin and C-peptide conjugated to Sepharose to improve its specificity. After adsorption of the antiserum, the cross-reactivities to insulin and C-peptide were each Since the direct assay sensitivity was not sufficient for measurement of endogenous hPI levels, a simple procedure for quantitative extraction of proinsulin-like material (PLM) from up to 40 ml of plasma on insulin antibody-Sepharose columns was developed. Log it-log slopes were calculated for dilutions of extracts of samples collected in the fasting state and 60 min after 75 g of oral glucose from eight healthy subjects. The slopes of 15 of the 16 samples did not differ significantly from the slope of the hPI standard. The fasting PLM concentration was 5.8 ± 3.0 fmol/ml (mean ± S.D, N = 8) and rose to 22.5 ± 6.7 fmol/ml (N = 6) 60 min after a 75-g oral glucose load. This assay will be useful in characterizing the metabolism and metabolic effects of biosynthetic human proinsulin and its intermediate forms and determining their therapeutic potential, and for measuring endogenous PLM in extracted plasma samples.
52 citations
TL;DR: Ability to increase proinsulin mRNA levels as an adaptation to pancreatectomy is indicated and is associated with the development of hyperglycemia, and the loss of this adaptation correlates with a worsening of glucose tolerance.
Abstract: These studies of partial pancreatectomy assess pancreatic proinsulin messenger RNA (mRNA) levels as an index of in vivo insulin biosynthesis, and show relationships to glucose homeostasis. Rats were subjected to sham operation, 50% pancreatectomy (Px), or 90% Px, and were examined after 1, 3, or 14 wk. Proinsulin mRNA was measured by dot hybridization to complementary DNA. After 50% Px there was a nearly complete adaptation of proinsulin mRNA. After 90% Px a marked increase of proinsulin mRNA occurred, but it was insufficient and it was not maintained with time. The deficit in insulin production is related to development of hyperglycemia. Sham-operated controls showed no worsening of fasting or fed blood glucose or of intraperitoneal glucose tolerance within the period of observation. Total proinsulin mRNA and pancreatic insulin content rose in proportion to body weight. 50% Px produced no change from controls in body weight or blood glucose. The concentration of proinsulin mRNA in the 50% pancreatic remnant paralleled that of controls after 1 and 3 wk, but then increased after 14 wk, such that total proinsulin mRNA approached control levels. This adaptive response was reflected by changes in serum insulin, but not by pancreatic insulin content, which was only 30% of control after 14 wk. Intraperitoneal glucose tolerance was impaired mildly, and did not worsen with time after pancreatectomy. 90% Px led to elevated fed blood glucose and reduced serum insulin after 3 wk, and fasting hyperglycemia was seen after 14 wk. Proinsulin mRNA concentration in the 10% pancreatic remnant showed an adaptive increase after 1 and 3 wk, such that total proinsulin mRNA reached 40% of control. After 14 wk, however, remnant proinsulin mRNA concentration was no longer increased; total proinsulin mRNA and pancreatic insulin content were severely reduced. Intraperitoneal glucose tolerance was impaired more dramatically than with the 50% Px animals, and worsened with time after operation. These observations indicate ability to increase proinsulin mRNA levels as an adaptation to pancreatectomy. Insufficiency of this adaptation is associated with the development of hyperglycemia, and the loss of this adaptation correlates with a worsening of glucose tolerance.
52 citations
TL;DR: A newly developed human hepatoma cell line, designated Hep G-2, expresses high-affinity insulin receptors meeting all the expected criteria for classic insulin receptors, and these receptors exhibit the negative cooperative interactions characteristic of insulin receptors in many other cell and tissue types.
Abstract: A newly developed human hepatoma cell line, designated Hep G-2, expresses high-affinity insulin receptors meeting all the expected criteria for classic insulin receptors. 125I-insulin binding is time-dependent and temperature-dependent and unlabeled insulin competes for the labeled hormone with a half-maximal displacement of 1-3 ng/ml. This indicates a Kd of about 10(-10) M. Since Scatchard analysis of the binding data results in a curvilinear plot and unlabeled insulin accelerates the dissociation of bound hormone, these receptors exhibit the negative cooperative interactions characteristic of insulin receptors in many other cell and tissue types. Proinsulin and des(Ala, Asp)-insulin compete for 125I-insulin binding with 4% and 2%, respectively, of the potency of insulin. Anti-(insulin receptor) antibody competes fully for insulin binding. The two insulin-like growth factors, multiplication-stimulating activity and IGF-I are 2% as potent as insulin against the Hep G-2 insulin receptor. Furthermore, Hep G-2 cells respond to insulin in several bioassays. Glucose uptake, glycogen synthase, uridine incorporation into RNA and acetate incorporation into lipid are all stimulated to varying degrees by physiological concentrations of insulin. In addition, these cells 'down-regulate' their insulin receptor, internalize 125I-insulin and degrade insulin in a manner similar to freshly isolated rodent hepatocytes. This is the first available human liver cell line in permanent culture in which both insulin receptors and biological responses have been carefully examined.
TL;DR: A significant correlation between mean insulin biosynthetic rates and mean proinsulin mRNA levels in 4-wk-old rats is demonstrated, suggesting that older, more hyperglycemic animals lose the capacity to increase rates of insulin biosynthesis and secretion.
Abstract: These studies of a rat model for non-insulin-dependent diabetes mellitus (NIDDM) were performed to determine whether hyperglycemia occurs when capacity to synthesize insulin is exceeded. The neonatal streptozocin (STZ)-treated rat has acute hyperglycemia with marked destruction of pancreatic β-cells, followed by gradual regeneration to 50–70% normal (β-cell number. At age 4 wk, fed serum glucose concentration is only mildly elevated relative to controls. With age, the rats become progressively hyperglycemic, and by 12 wk they have marked impairment of glucose-stimulated insulin release. In these studies, dexamethasone (0.125 mg/kg/day for 4 days) was administered to control and to STZ-treated animals to produce insulin resistance. The relationship between insulin biosynthesis and serum glucose concentrations was assessed. In control rats, response to dexamethasone was similar at both 4 and 12 wk. Serum glucose levels and pancreatic insulin concentration remained unchanged. Both insulin biosynthetic rates (as measured by 3 Hleucine incorporation into proinsulin) and proinsulin mRNA levels increased twofold. STZ-treated rats at age 4 wk demonstrated mild hyperglycemia. Dexamethasone injection resulted in an increase in insulin biosynthesis and proinsulin mRNA in these animals, while serum glucose did not increase. STZ-treated rats at 12 wk showed more profound hyperglycemia (serum glucose 315 ± 38 mg/dl versus control, 187 ± 12 mg/dl). A marked rise in serum glucose (to 519 ± 42 mg/dl) was observed after4 days of dexamethasone injection. Pancreatic insulin content became severely depleted relative to salineinjected, STZ-treated animals, and there was no response of levels of proinsulin mRNA. These studies demonstrate a significant correlation between mean insulin biosynthetic rates and mean proinsulin mRNA levels in 4-wk-old rats. In these rats with mild glucose intolerance related to a decrease in insulin synthesis, the rate of biosynthesis can still increase if demand for insulin is acutely increased by induction of an insulin-resistant state. If the correlation between proinsulin mRNA and biosynthesis exists in 12-wk-old animals, the data suggest that older, more hyperglycemic animals lose the capacity to increase rates of insulin biosynthesis and secretion; severe hyperglycemia ensues when capacity to synthesize insulin is exceeded.
TL;DR: Islets of rats with NIDD, once removed from the chronic in vivo exposure to diabetic metabolic disorders, can behave as isolated islets of normal rats, at least as far as insulin handling is concerned.
Abstract: Non-insulin dependent diabetes (NIDD) was obtained in adult rats after a neonatal streptozotocin injection. In the fed state 3- to 5-month-old rats with NIDD exhibited modestly elevated plasma glucose levels (controls, 131 +/- 7 mg/dl; diabetics, 159 +/- 4 mg/dl; P less than 0.01), impaired glucose tolerance, and a very low insulin response after glucose injection. In the present study the secretion and biosynthesis of insulin were measured using isolated islets from these rats. The insulin and DNA content of islets freshly isolated from rats with NIDD were significantly lower (60% and 80%, respectively, P less than 0.001) than in the controls. The insulin content per islet cell from diabetic rats was also significantly reduced (75%) (P less than 0.01) as compared to controls. At a low glucose concentration (2.8 mM) the insulin release from islets of diabetic rats was 60% (P less than 0.05) of that the controls. At a glucose concentration of 16.5 mM it was stimulated 5-fold from the islets of NIDD rats and 7-fold from the islets of control rats. (Pro)insulin biosynthesis, assessed in the same islets by measuring the incorporation of [3H]phenylalanine into immunoprecipitable material, was significantly higher (50%) (P less than 0.01) in islets from NIDD rats when measured at 2.8 mM glucose. Although (pro)insulin biosynthesis in these islets was significantly stimulated by 16.5 mM glucose (5-fold), it was less (P less than 0.05) than in the control islet (9-fold). To determine whether the derangements described above in the islets of the rats with NIDD could be modified by changing the environmental conditions of the B cells, corresponding experiments were performed after a 5-day culture of the islets at 5.5 mM glucose or at 11 mM glucose. The insulin release and the (pro)insulin biosynthesis, either measured in basal or stimulated states, were then found to be similar in the islets of diabetic and control islets after the 5.5 mM glucose culture period. By contrast, after the 11 mM glucose culture period the insulin release and the proinsulin biosynthesis in the islets of diabetic rats were found significantly less stimulated by 16.5 mM glucose than in the control islets. This suggests that islets of rats with NIDD, once removed from the chronic in vivo exposure to diabetic metabolic disorders, can behave as isolated islets of normal rats, at least as far as insulin handling is concerned.(ABSTRACT TRUNCATED AT 400 WORDS)
TL;DR: Thyrotoxic subjects showed normal basal and peak levels of serum immunoreactive insulin and serum C-peptide, and thyroid hormone excess appears to affect adipocyte insulin receptor number directly, but change in receptor number is not associated with change in adipocytes insulin sensitivity in hyperthyroidism.
Abstract: The pattern of insulin secretion following an oral glucose load and the insulin receptor status and insulin sensitivity of adipocytes have been studied in patients with thyrotoxicosis and in matched controls. Thyrotoxic subjects showed normal basal and peak levels of serum immunoreactive insulin (peak, 69.0 +/- 6.8 vs 54.3 +/- 8.8 mU/l) and serum C-peptide (peak, 1.95 +/- 0.13 vs 1.71 +/- 0.12 nmol/l for thyrotoxic and control subjects, respectively). Peak serum proinsulin was higher in the thyrotoxic group (64.8 +/- 7.3 vs 39.0 +/- 3.7 pmol/l; P less than 0.01). Maximum specific insulin binding to adipocytes was decreased in the thyrotoxic group (1.80 +/- 0.18 vs 2.62 +/- 0.27%; P less than 0.025) and half-maximum displacement of tracer insulin was similar in the two groups, suggesting that reduced receptor number rather than reduced affinity accounted for the difference. However, adipocyte insulin sensitivity was normal as judged by half-maximal stimulation values of 13.9 +/- 3.6 vs 11.4 +/- 2.1 pmol/l, respectively for lipogenesis and 24.3 +/- 2.2 vs 24.6 +/- 3.6 pmol/l, respectively for glucose transport. Hence, thyroid hormone excess appears to affect adipocyte insulin receptor number directly, but change in receptor number is not associated with change in adipocyte insulin sensitivity in hyperthyroidism. The normal insulin secretion together with the failure to demonstrate abnormal insulin sensitivity of one of the major peripheral tissues suggests that disturbed hepatic rather than peripheral insulin responsiveness may be responsible for the glucose intolerance of hyperthyroidism.
TL;DR: The data suggest that insulin, at pharmacological doses, interferes with embryo development through a non-metabolic pathway, probably via a growth-type receptor.
Abstract: The teratogenic effect of insulin in early vertebrate embryos is controversial and the mechanisms involved are unknown. We studied the effects of pharmacological doses of insulin in chick embryos during the period of differentiation. We compared the effects of insulin with two proinsulins, desoctapeptide-insulin and multiplication-stimulating activity, peptides that have little insulin-like metabolic activity while they have significant growth effects. Chick embryos at 46 h of development were injected with the different peptides. At 96 h the mortality and abnormal growth elicited by the peptides were dose-dependent. Considering the indices of lethality (LD50) and affected embryos (ED50) as 100% for insulin, proinsulin was 59–66% as potent as insulin, desoctapeptide-insulin 2–6% and multiplication-stimulating activity 176–204%. In the surviving embryos, insulin (5 μg, decreased DNA, RNA and protein content by 49%, 40% and 48% respectively compared with controls. The effects of insulin were not corrected by simultaneous glucose injections. These data suggest that insulin, at pharmacological doses, interferes with embryo development through a non-metabolic pathway, probably via a growth-type receptor.
TL;DR: It was concluded that the A-chain loop is exposed on the surface of proinsulin, while the C-terminal B-chain is not available for binding, and monoclonal antibodies are suggested to use for the screening of atypical insulins in the serum of diabetic patients and for the further refinement of insulin and Proinsulin measurements.
Abstract: High-affinity monoclonal antibodies (MAB) were obtained from lymph node cell fusions. Affinities ranging from 0.8 × 10 9 L/M to 5.2 ×109 L/M were calculated from binding studies with monoiodinated human, bovine, and porcine insulins and human proinsulin. Two monoclonal antibodies were specific for human insulin, recognizing an epitope involving the amino acid B-30 (Thr). Another two monoclonal antibodies were bound to the C-terminal end of the B-chain near B-30. The B-chain-specific monoclonal antibodies did not bind human proinsulin. One monoclonal antibody recognized the A-chain loop in the positions A-8 to A-10. This antibody bound also to human proinsulin. It was concluded that the A-chain loop is exposed on the surfaceof proinsulin, while the C-terminal B-chain is not available for binding. The study shows that monoclonal antibodies can be used to characterize structures of insulin and proinsulin. In contrast to x-ray studies, the molecules can be used at low concentrations in soluble form. It is suggested to use monoclonal antibodies for the screening of atypical insulins in the serum of diabetic patients and for the further refinement of insulin and proinsulin measurements.
TL;DR: Treatment of biosynthetic Proinsulin with trypsin under mild conditions led to a decrease in proinsulin-like immunoreactivity concomitant with an increase in C-peptide and insulin-like Immunoreactivity, indicating that the proins insulin-specific antiserum did not preferentially recognize intermediates of proinsulus cleavage.
Abstract: Antibodies have been raised against biosynthetic human proinsulin that show less than 1% cross-reactivity with human insulin and C-peptide. A sensitive (IC50 0.16 pmol/ml; minimum detectable concentration 0.004 pmol/ml) radioimmunoassay has been developed using this antiserum and 125I-proinsulin that will measure proinsulin-like immunoreactivity in human serum without the need for prior separation of insulin or C-peptide. In healthy, fasted subjects (N = 23), the serum proinsulin concentration was 0.015 +/- 0.001 pmol/ml (mean +/- SEM). In six healthy subjects, serum proinsulin rose to 250% of basal after 120 min in response to 100 g oral carbohydrate, but to only 130% after 60 min following 25 g oral carbohydrate. The proinsulin/total immunoreactive insulin ratio and the proinsulin/C-peptide ratio fell sharply after both high and low carbohydrate loads. Endogenous human serum proinsulin-like immunoreactivity released into the circulation after 100 g carbohydrate was eluted from a Mono Q high-performance, ion-exchange column with the same retention time as biosynthetic human proinsulin. Treatment of biosynthetic proinsulin with trypsin under mild conditions led to a decrease in proinsulin-like immunoreactivity concomitant with an increase in C-peptide and insulin-like immunoreactivity, indicating that the proinsulin-specific antiserum did not preferentially recognize intermediates of proinsulin cleavage.
TL;DR: The construction of a plasmid vector, pVT25, which allows an efficient and direct selection for transformed cells carrying recombinant plasmids is described, and the production of the characteristic C-peptide of proinsulin, monitored by radioimmunoassay, was shown to be under the control of the inducible promoter of the colE3 gene.
TL;DR: The methods described in this chapter yield IGF of high purity and with high recovery within approximately 25 workdays.
Abstract: Publisher Summary Insulin-like growth factors (IGFs) I and II are the designations for two polypeptides of approximately 7.5 kDa isolated from human serum whose amino acid sequences are homologous to proinsulin. In vitro , both factors show insulin-like metabolic effects on adipose and muscle cells and mitogenic activity on various cells, especially chondroblasts and fibroblasts. In vivo , IGF I promote the growth of hypophysectomized animals, and its serum concentration is regulated by growth hormone. IGF I is identical to somatomedin C, which mediates the growth effects of growth hormone. Multiplication stimulating activity (MSA) is the rat homolog to human IGF II. Originally, IGF I and II were isolated from an acid ethanol extract of a plasma fraction (precipitate B). The methods described in this chapter yield IGF of high purity and with high recovery within approximately 25 workdays.
TL;DR: The application of Fast Atom Bombardment Mass Spectrometry to the measurement of small differences in molecular weight is discussed in relation to the spectra of bovine proinsulin and a genetic variant.
TL;DR: The purified thiol peptidase has the highest affinity/lowest efficiency for insulin, an intermediate affinity/intermediate efficiency for B chain of insulin and the lowest affinity/highest efficiency for glucagon.
Journal Article•
TL;DR: It is demonstrated that glucose modulates selectively the level of proinsulin mRNA during incubation of isolated pancreatic islets and that changes in thelevel of pro Insulin mRNA play an important role in regulation of insulin biosynthesis.
Abstract: Isolated rat pancreatic islets were incubated in high (28 mM) or low (2.8 mM) glucose for 0, 1 or 4 hr and insulin biosynthesis and messenger RNA quantified. During the 4 hr course of the experiments the fraction of protein synthesised specifically into proinsulin increased in the presence of high vs low glucose (14.3 +/- 3.7 vs 0.8 +/- 0.5%, p less than 0.01). The relative amount of proinsulin mRNA was found to be about 5-fold higher in islets incubated in high glucose at 4 hr as determined by RNA blot hybridization. Total translatable islet mRNA was unaffected by glucose in the medium. These data extend observations of changes in proinsulin mRNA in rats in vivo during fasting and glucose injection, and suggest a direct effect of glucose on islets. These data further demonstrate that glucose modulates selectively the level of proinsulin mRNA during incubation of isolated pancreatic islets and that changes in the level of proinsulin mRNA play an important role in regulation of insulin biosynthesis.
TL;DR: An insulin-like hybrid molecule consisting of the A chain of insulin and a B chain corresponding to the B domain of human insulin- like growth factor I (growth factor I sequence 1-30) has been synthesized essentially by the procedures developed in this laboratory for the synthesis of insulinand analogues.
Abstract: An insulin-like hybrid molecule consisting of the A chain of insulin and a B chain corresponding to the B domain of human insulin-like growth factor I (growth factor I sequence 1-30) has been synthesized essentially by the procedures developed in this laboratory for the synthesis of insulin and analogues. The hybrid competed with 125I-insulin for insulin receptors in rat liver plasma membranes and was a full agonist in stimulating incorporation of [3(-3)H]glucose into lipids in rat adipocytes. In both assays, the compound displayed ca. 2% of the potency of insulin. The compound was recognized by anti-insulin antibodies but was only ca. 0.25% as potent as insulin in this activity. The hybrid exhibited growth-promoting activity in fibroblasts, displaying 3-8% of the activity of insulin. In contrast, the compound was recognized by insulin-like growth factor carrier proteins, a property not associated with insulin. Two points of nonhomology between the B chain of insulin and the B domain of insulin-like growth factor I are considered in connection with these observations.
TL;DR: The fingerprint method allows, for example, the simultaneous analysis of porcine and human insulin, the identification of a modified insulin generated in dosing devices, as well as the individual analysis of the two disulfide linkages between the A- and B-chain in refolded insulins.
Abstract: Proteolysis of insulin or (pre)proinsulin with S. aureus protease V8 in Tris buffer at neutral pH yields a characteristic pattern of peptide fragments that is resolved using high-performance liquid chromatography. Identification of the fragments of interest was achieved by comparison of insulins of different species, of modified insulins and of proinsulin and N-extended proinsulin, and by amino acid analysis. The fingerprint method allows, for example, the simultaneous analysis of porcine and human insulin, the identification of a modified insulin generated in dosing devices, as well as the individual analysis of the two disulfide linkages between the A- and B-chain in refolded insulins.
TL;DR: Three monoclonal antibodies to insulin have been tested for cross-reactivity with 11 species variants of insulin and three of proinsulin allowing the identification of two epitopes recognized by the MAb which encompass the regions in the A- and B-chains of insulin subject to frequent evolutionary amino acid substitutions.
TL;DR: It is concluded that bovine insulin purified to less than 1 ppm proinsulin is significantly less immunogenic than its conventional Proinsulin contaminated counterpart but even at this level of purity is still more immunogenicthan human insulin of equivalent purity.
Abstract: Twenty-six Type 1 diabetic patients previously treated for 10-20 months with twice daily conventional bovine isophane insulin (containing at least 1000 ppm proinsulin) were changed to highly purified (less than 1 ppm proinsulin) bovine isophane for 6 months (Switch group). Insulin antibody levels fell significantly from a geometric mean of 14.9 to 9.1 micrograms/l. Thirty-two patients with newly diagnosed Type 1 diabetes were treated with the same highly purified bovine isophane insulin twice daily for 6 months (Starter group). Their insulin antibody levels rose from a geometric mean of 1.9 to 8.2 micrograms/l in contrast to values of 1.4 rising to 16.3 micrograms/l in an age and sex matched historical control group treated from diagnosis only with twice daily conventional bovine isophane insulin. Lipoatrophy at injection sites developed in three (9%) in the Starter group treated with highly purified bovine isophane compared to 7 (22%) of those on conventional bovine isophane. Insulin dose and diabetic control did not differ between the groups. Starter and Switch groups were subsequently treated with semi-synthetic human isophane insulin for 6 months during which insulin antibody levels fell significantly from a geometric mean of 8.5 to 4.4 micrograms/l (p less than 0.001). We conclude that bovine insulin purified to less than 1 ppm proinsulin is significantly less immunogenic than its conventional proinsulin contaminated counterpart but even at this level of purity is still more immunogenic than human insulin of equivalent purity.
TL;DR: Highly active insulin-degrading activity was found using cell suspensions of 22 cloned and 8 subcloned cell lines derived from RINm as well as 11 other continuous cell linesderived from a variety of nonislet tissues of rat, mouse, and human origin.
Abstract: RIN-m cells, cultured from a rat insulinoma, not only bind and secrete but also degrade insulin (Diabetes 1982; 31:521–31). The insulin-degrading activity resides in the cytosol and is similar to the insulin-specific proteases previously described in muscle and other tissues. It has an apparent Km of 0.15 μM for porcine insulin in crude cell-free extracts, a competitive inhibition constant for proinsulin that is close to the Km, and a lower but measurable affinity for glucagon. The enzyme is inactive at pHs below 6.0, indicating that it is not lysosomal, is completely inhibited by Nethylmaleimide, and exhibits apparent competitive inhibition constants (μM) for the following peptides: desoctapeptide insulin, 0.043; guinea pig insulin, 0.048; proinsulin, 0.64; insulin B-chain, 1.17; glucagon, 7.0; and cyclic somatostatin, 8.6. Highly active insulin-degrading activity was found using cell suspensions of 22 cloned and 8 subcloned cell lines derived from RINm as well as 11 other continuous cell lines derived from a variety of nonislet tissues of rat, mouse, and human origin. Homogenates of the original rat islet tumor and cytosol of normal rat islets also contained insulin-degrading activity. Although insulin protease is present in a variety of tissues, it may have an additional regulatory function in cells that are actively synthesizing, storing, and secreting insulin.
TL;DR: It is concluded that a covalent aggregate of therapeutic insulin circulates in diabetic patients who use a variety of insulin preparations, and these aggregates may promote the formation of anti-insulin antibodies and affect the action of insulin.
Abstract: The tendency of insulin in high concentrations to selfassociate and the widespread presence of insulindegrading enzymes suggest that fragments and/or aggregates of insulin may circulate in normal and insulin-dependent diabetic (IDOM) individuals. To examine this possibility, we have analyzed, by sensitive physicochemical methods, immunoreactive insulin (IRI) taken from the blood of 9 healthy volunteers and 12 insulin-dependent diabetic patients. IRI from the blood of the normal volunteers was composed of 6000 (91.0 ± 1.4%) and 9000 (9.0 ± 1.4%) molecular weight (mol wt) material. By 10% polyacrylamide disc gel electrophoresis (PAGE) and reverse-phase, high-performance liquid chromatography (HPLC), the 6000 mol wt material was indistinguishable from human insulin standards and insulin fragments were not found. C-peptide reactivity in the 9000 mol wt material confirmed the expected presence of proinsulin and intermediates of proinsulin conversion. IRI harvested from the blood of 12 C-peptide-negative IDDMs, using a variety of insulin preparations, also separated into 6000 (80.5 ± 3.9%) and 9000–12,000 (19.5 ± 3.9%) mol wt material. By HPLC, 6000 mol wt IRI was either pork insulin (in volunteers using pure pork insulin) or a mixture of beef (∼90%), pork (∼10%) and deamidated beef (trace) insulin in those using a beef-pork mixture. However, the 9000–12,000 mol wt material had characteristics entirely distinct from proinsulin of either human or animal origin: (1) C-peptide reactivity was undetectable using any of three sensitive radioimmunoassay systems, (2) on PAGE it migrated more rapidly than proinsulin-like material, and (3) in contrast to proinsulin, it was unaffected by proteolytic degradation. We conclude that a covalent aggregate of therapeutic insulin circulates in diabetic patients who use a variety of insulin preparations. Insulin aggregates may account for a relatively large amount of the total IRI in IDDMs, and these aggregates may promote the formation of anti-insulin antibodies and affect the action of insulin.
TL;DR: Short-term effects of human proinsulin on metabolic rates and its long-term action on enzyme induction were studied in primary cultures of rat hepatocytes and in the perfused rat liver, and compared with the effects of bovine insulin.
Abstract: Short-term effects of human proinsulin on metabolic rates and its long-term action on enzyme induction were studied in primary cultures of rat hepatocytes and in the perfused rat liver, and compared with the effects of bovine insulin. In the perfused rat liver, proinsulin decreased the glucagon-dependent increase of glycogenolysis. The action of 0.5 nM glucagon was almost completely suppressed by 100 nM proinsulin. Proinsulin and insulin showed similar potency. In cultured rat hepatocytes, proinsulin stimulated glycolysis up to fivefold with a half-maximal effective dose of 30 nM. Proinsulin induced the key glycolytic enzymes glucokinase and pyruvate kinase by twofold and antagonized the glucagon-dependent induction of phosphoenolpyruvate carboxykinase with a halfmaximal effective dose at 3 nM. For the effects in cultured hepatocytes, about 100-fold higher concentrations of proinsulin than of insulin were required.
TL;DR: In this article, the authors used restriction fragment length polymorphisms adjacent to the insulin gene to examine cosegregation with hyperproinsulinemia, and demonstrated linkage of hyperproinulininemia and the insulin genes in this family with a LOD score of 1.8.
Abstract: Two previously described pedigrees with familial hyperproinsulinemia have elevated proinsulin conversion intermediates resulting from amino acid substitutions in the proinsulin molecule. In contrast, a third family with elevated levels of an apparently normal proinsulin molecule may have a defect in the converting process. To determine if the defect in this family lies in the insulin gene region, we used restriction fragment length polymorphisms adjacent to the insulin gene to examine cosegregation with hyperproinsulinemia. We demonstrate linkage of hyperproinsulinemia and the insulin gene in this family with a LOD score of 1.8, suggesting that the defect lies in or near the insulin gene. This method has wide applicability in determining whether hyperproinsulinemia or hyperinsulinemia is the result of defects at the insulin gene, and should permit the detection of new defects at or near this locus.