Showing papers on "Proinsulin published in 1973"

Metabolism of Proinsulin, Insulin, and C-Peptide in the Rat

Abstract: The renal extraction and excretion of bovine proinsulin, insulin, and C-peptide and the contribution of the kidney to their total metabolic clearance rate (MCR) were studied in the rat. Metabolic clearance rates were measured by the constant infusion technique and plasma and urine concentrations of each polypeptide were determined by radioimmunoassay. The MCR of insulin (16.4+/-0.4 ml/min) was significantly greater than that of either proinsulin (6.7+/-0.3 ml/min) or C-peptide (4.6+/-0.2 ml/min). Metabolic clearance rates were independent of plasma levels over a range of steady-state plasma concentrations varying from 1 to 15 ng/ml.In contrast to the differences in their metabolic clearance rates, the renal disposition of the three polypeptides was similar, being characterized by high extraction and very low urinary clearance. The renal arteriovenous difference of proinsulin, insulin, and C-peptide averaged 36, 40, and 44%, respectively, and was linearly related to their arterial concentration between 2 and 25 ng/ml. When glomerular filtration was markedly reduced or stopped by ureteral obstruction, the renal extraction of proinsulin, insulin, and C-peptide was invariably greater than the simultaneously measured extraction of inulin, indicating that these polypeptides are removed from the renal circulation by both glomerular filtration and direct uptake from peritubular capillary blood. The fractional urinary clearance of each polypeptide never exceeded 0.6%, indicating that more than 99% of the amount filtered was sequestered in the kidney. The renal removal of proinsulin and C-peptide from the circulation accounts for 55 and 69% of their metabolic clerance rates, while the renal contribution to the peripheral metabolism of insulin was smaller, averaging 33%. This difference is due to the fact that insulin, but not the other two polypeptides, is metabolized to a significant extent by the liver. These results define the renal handling of proinsulin, insulin, and C-peptide in the rat and indicate that in this species the kidney represents a major site for insulin metabolism and is the main organ responsible for the degradation of proinsulin and C-peptide.

A comparative study on the metabolism of human insulin and porcine proinsulin in man.

Abstract: SUMMARY 1. The metabolism of unlabelled monocomponent human insulin and porcine proinsulin was studied in ten normal subjects (five males and five females) by using a priming dose-constant-infusion technique. In each subject, the metabolic clearance rate (MCR) was measured at four separate steady-state hormone concentrations averaging 16-21 6 punitslml (insulin) and 4-2-42.8 ng/ml (proinsulin). 2. For insulin the MCR fell progressively from 34 ml kg-' min-' at a mean fasting insulin concentration of 3.8 punitslml to 11.4 ml kg-' min-' at the highest concentration achieved (280 punits/ml); for proinsulin MCR averaged 3.7 ml kg - ' min-' at a mean plasma concentration of 4.2 ng/ml and fell to 2.71 ml kg-' min-' at 10.7 ng/ml, remaining constant thereafter at concentrations up to 71 ng/ml. 3. The half-disappearance time (T+) from the plasma, after the end of the infusion, averaged 4.3 min for insulin and 25.6 min for proinsulin. 4. The apparent distribution space (DS) was similar for both hormones (83 ml/kg of insulin and 98.9 ml/kg of proinsulin). 5. There was a direct correlation between T+ and DS for both hormones. 6. Although the higher MCR of insulin was reflected in its shorter T+, there was, for each hormone, no relationship between MCR and T+. 7. The biological potency of porcine proinsulin, as judged by its effect on plasma glucose, was approximately 5% of that of insulin. 8. The responses of serum growth hormone and cortisol were shown to be directly related to the degree of hypoglycaemia induced.

Insulin in Invertebrates and Cyclostomes

Abstract: It seems increasingly clear that insulin is a hormone that does not occur exclusively in vertebrates. Several independent reports now exist giving evidence of insulin production in the digestive tissues of both deuterostomian and protostomian invertebrates. Cells with some light-microscopical and ultrastructural characteristics of vertebrate B-cells have also been observed. Recently, evidence has been obtained that insulin can act as a hypoglycemic hormone, promoting glycogen synthesis, also in a protostomian invertebrate, the gastropod mollusc, Strophocheilus oblongus . The endocrine pancreas of the cyclostomes occupies a key position in the comparative endocrinology of the islet parenchyma and in the evolution of insulin production. It may represent an evolutionary link between the presumably gut-connected dispersed insulin-producing cells of deuterostomian invertebrates and the pancreatic islets of gnathostomian vertebrates. This hypothesis was supported by the fact that cells with light-microscopical and ultrastructural similarities to the islet B-cells were observed in the bile duct mucosa of the hagfish, Myxine glutinosa . However, immunofluorescent studies with antisera against human insulin and C-peptide did not show any immunoreactive material outside the B-cells of the endocrine pancreas. Particular attention was paid to elucidate the biological significance of the large cystic cavities that are so typical for the cyclostomian islet parenchyma. The working hypothesis that they may contain stored insulin, proinsulin (or even “proto-proinsulin”) was not supported by immunofluorescence, autoradiographic, or ultrastructural investigations, nor by proinsulin assays. It is possible that hagfish islet B-cells contain zinc, despite the fact that the amino acid residue in B10-position is aspartic acid instead of histidine. The biosynthesis of hagfish insulin shows a pattern similar to that in gnathostomes. Its rate is related to the ambient temperature and at 11 C the conversion of proinsulin to insulin lasts several days.

The stimulus-secretion coupling of glucose-induced insulin release. XIV. Glucose regulation of insular biosynthetic activity.

Abstract: The biosynthetic activity of the pancreatic β-cell was assessed by measuring the incorporation of labeled leucine in rat isolated islets, both immunological and chromatographic procedures being used to characterize the newly synthesized peptides. The conversion rate of proinsulin and the release of newly synthesized hormonal peptides were also evaluated. Glucose and rnannose, unlike galactose, fructose, ribose and xylitol stimulated (pro)insulin biosynthesis. A sigmoidal curve characterized the relationship between (pro)insulin synthesis and the glucose concentration of the incubation medium, the treshold value for the stimulant action of glucose being lower for insulin synthesis than for insulin release. Glucose preferentially stimulated the synthesis of hormonal peptides as distinct from that of other insular proteins. This preferential action was suppressed by mannoheptulose and 2-deoxyglucose, but unaffected by cycloheximide or dinitrophenol. Based on these observations, a model is proposed which coul...

Microtubules and beta cell secretion.

Abstract: Publisher Summary This chapter presents the concept of the involvement of the microtubular-microfilamentous system in the intracellular transport of beta granules and the mechanism by which insulin is released from the beta cell. Glucose stimulates both the release and the synthesis of insulin in beta cells. Insulin release occurs within a few seconds after stimulation with glucose. The sequence of events in the formation of insulin are as follows: (1) proinsulin is formed within the endoplasmic reticulum and is transferred by an energy-requiring process to the Golgi complex, (2) distinct beta granules form within the Golgi complex and are pinched off into the cytoplasm, (3) the C peptide of proinsulin is removed in the Golgi area, and (4) zinc binds with the insulin in the mature beta granules, resulting in the formation of a crystalline matrix. Thus, the storage form of insulin in beta cells is the beta granule that resembles a microcrystal of zinc insulin. The beta granule is surrounded by a smooth membranous sac with a space between the granule and the encompassing membrane. Biochemical studies on microtubular protein isolated from the brain indicate that these structures contain an actin-like substance.

Cocrystallization of Proinsulin and Insulin

Abstract: THE occurrence of small amounts of proinsulin in crystalline preparations of bovine, porcine, and rat insulin1–4 and the many similarities in properties of proinsulin and insulin prompted us to consider the possibility that proinsulin co-crystallizes with insulin. Spectral5 as well as immunological6 studies have provided evidence that the insulin moiety in proinsulin has a very similar conformation to that of insulin, and proinsulin exhibits the same tendency to form dimers in acidic solution and higher polymers, mainly hexamers, in neutral solutions containing zinc ions7. Studies on crystals of proinsulin by Low and coworkers8 have confirmed the presence of dimers in the unit cells of these crystals. All these observations suggest that the connecting polypeptide segment in proinsulin is oriented away from those surfaces involved in dimer and hexamer formation.

Dynamic Synthesis and Release of Insulin and Proinsulin from Perifused Islets

Abstract: Insulin and proinsulin syntheses from leucine-H-3 were studied in glucose-stimulated perifused rat islets, with particular attention being placed on the contribution of de novo synthesis to the characteristic second phase of insulin release At fifty to sixty minutes, when the second phase had reached approximately steady state, most of the islet radioactivity was still in the proinsulin fraction; newly synthesized insulin during this interval represented less than 05 per cent of the total insulin secreted By 110 to 120 minutes, this value had increased, but to less than 175 per cent The half time of conversion of proinsulin to insulin was approximately sixty minutes At either fifty to sixty or 110 to 120 minutes, proinsulin was less than 5 per cent of the total hormone secreted The ratio of specific activities for secreted to islet insulin was less than unity at sixty minutes and exceeded unity at 120 minutes; the comparable ratios for proinsulin were somewhat above unity at both times Results suggest newly synthesized insulin is not responsible for the second phase of insulin release In addition, storage of insulin is nonhomogeneous with preferential release of the older nonlabeled hormone occurring during the first hour of glucose stimulation

In vitro studies of the rate of proinsulin and insulin turnover in seven human insulinomas.

Abstract: Summary. Human insulinoma tissue excised from seven patients was incubated with glucose (3.0 mg/ml) and 3H-leucine (100 μCi/ml) for a 15 min. pulse period. One third of the tissue was homogenized immediately in 0.3 M sucrose (pH 6.0), one third was homogenized after a 20 min. chase (with L-leueine 37S μg/ml) and the remaining third after an 80 min. chase. Subcellular fractions (cell debris, mitochondria, secretory granules, microsomes and S-100) were prepared by centrifugation. Their identity was confirmed by ultrastructural examination. Since only tumours revealing typical β-granules were investigated the secretory granule fractions contained the same types of granules as found in the normal human B-cell.—The S-100 contained approximately 40% of the immuno-reactive insulin (IRI). The secretory granule samples from five tumours were fractionated by sucrose gradient ultracentrifugation. At the three time intervals examined, a peak of radioactivity and IRI was located at 1.60 M sucrose in three of the tumours. The different granule types were not separable in the sucrose gradients. Endogenous proinsulin-like components (proinsulin) and insulin were released throughout the incubation. During incubation of tumour tissue the ratio of IRI release to content was significantly higher than that from isolated human pancreatic islets. Newly synthesized proinsulin was detected in the medium after the 15 min. pulse in five of the tumours; radioactive proinsulin and insulin were present in all the media after the 20 and 80 min. chase periods. Islets isolated from the pancreatic tissue of two insulinoma patients showed a much slower proinsulin and insulin turnover.—These data support the conjecture that human insulinomas have a more rapid turnover of proinsulin and insulin than normal pancreatic islet tissue, apparently as the consequence of defective IRI storage and release mechanisms.

Studies on the conversion of proinsulin to insulin in the isolated islets of Langerhans in the rat.

Abstract: A time course study on proinsulin and insulin biosynthesis was carried out, and the results showed that proinsulin was synthesized in the microsomes and that conversion of proinsulin took place in the granule fraction (Golgi complex and granules) in isolated rat islets. When the granule fraction was incubated with exogenous 3H-labelled proinsulin for 1.5 h, no conversion of labelled proinsulin was observed. Nevertheless pulse-chase experiments showed that endogenous 3H-labelled proinsulin was converted to insulin in the granule fraction. Granular membrane and granular medium were prepared according to the method of Viveros. When endogenous 3H-labelled proinsulin was incubated with these fractions the conversion of labelled proinsulin was found only in the presence of the granular membrane fraction. The percentages of proinsulin and insulin in the total of proinsulin plus insulin changed from 80.6% and 19.4% before the incubation to 65.0% and 35.0% after the incubation. The conversion of exogenous 3H-label...

Comparative Aspects of Proinsulin and Insulin Structure and Biosynthesis

Abstract: This review summarizes currently available information on the composition and structure of vertebrate insulins and proinsulins. Consideration is given to the important structural features of insulin and its precursor that are involved in the function and formation of the active hormone. Studies on the biosynthesis of insulin in teleost fishes indicate the existence of larger single chain precursor forms similar to the mammalian proinsulins. Preliminary results of experiments on insulin biosynthesis in the hagfish ( Myxine glutinosa ), which has the most primitive islet parenchyma of all vertebrates, indicate the existence of a similar biosynthetic mechanism. The major storage product in the B-cells in all the vertebrate species studies thus far is insulin rather than proinsulin. In fishes an intracellular tryspin-like enzyme may suffice to convert proinsulin to insulin, while in mammals a more complex mechanism involving both an endopeptidase and an exopeptidase is probably required. Conversion occurs within the Golgi apparatus and newly formed secretory granules in the B-cells. The similarity to the higher vertebrates in the biosynthesis and molecular structure of insulin in the primitive hagfish indicates that the properties and biological role of this hormone have remained fairly constant throughout several hundred million years, or that its evolution has followed the same pattern in most extant organisms despite considerable differences in their origin and living conditions. A hypothesis for the evolution of insulin and of the B-cells based on the biosynthetic mechanism involving proinsulin and its conversion to insulin is briefly considered.

Insulin and proinsulin content of pancreases from diabetic and nondiabetic subjects.

Abstract: The content of immunoreactive insulin (IRI) extractable with acid ethanol from the head, body and tail of the pancreas was. studied in thirty-two nondiabetics, eleven maturity-onset diabetics and four juvenile diabetics. In nondiabetics IRI content ranged from 0.1 to 4.8 U. per gram wet pancreas. The tail had the highest insulin content, but the body of the pancreas could be taken to represent the entire pancreas. The insulin content of pancreases from eleven maturity-onset diabetics ranged from 0.1 to 6.5 U. per gram wet pancreas. It showed a progressive decline with the duration of diabetes. The IRI content of pancreases from four juvenile diabetics was low. The proportion of proinsulinlike component, estimated by gel filtration in the pancreatic extracts, ranged from 0.2 to 5 per cent of the insulin peak and was not different in diabetics.

Stimulation of H-3-leucine incorporation into the proinsulin and insulin fraction of isolated pancreatic mouse islets in the presence of glucagon, theophylline and cyclic AMP.

Abstract: Incorporation of H-3-leucine into proinsulin and insulin and insulin secretion were determined in isolated pancreatic islets of mice. Batches of forty islets were incubated for three hours at 0, 100 or 300 mg. per cent glucose alone or in the presence of glucagon, theophylline or cyclic AMP. Proinsulin and insulin were separated by gel filtration on columns of Sephadex C50fine in 1 M. acetic acid. In the absence of glucose, no stimulation of insulin biosynthesis as judged from the incorporation of H-3-leucine was observed with glucagon, cyclic AMP or dibutyryl cyclic AMP; insulin, secretion was not significantly stimulated. At 100 and 300 mg. per cent glucose, both leucine incorporation and insulin secretion were potentiated by the substances tested; the effect of glucagon was pronounced mainly at high concentrations of glucose. However, leucine incorporation and insulin secretion were not stimulated to the same extent in all cases. Biosynthesis of insulin thus appears to be not always directly linked to insulin secretion. It is suggested that glucagon and the adenylcyclase system, besides regulating insulin secretion, are involved in insulin biosynthesis.

Proinsulin-Specific Antibodies in Human Sera

Abstract: Sera from nine diabetic patients treated with commercial insulin contained antibodies which reacted to I-125-bovine proinsulin. These antibodies had two distinct components: anti-insulin antibodies which crossreacted with proinsulin, and antiproinsulin antibodies, which were specific to proinsulin and did not react with insulin (purified, single component). Proinsulin-specific antibodies were detectable by their binding to I-125-bovine proinsulin in sera adsorbed with insulin-phe-sepharose or in sera preincubated with excess insulin. I-125-bovine pro insulin-reactive antibodies showed species specificity. They reacted weakly with porcine proinsulin and appeared to have no crossreactivity with porcine-C-peptide. The data suggest that proinsulin-specific antibodies (a) may be induced by the contaminants present in commercial insulin; (b) do not inactivate exogenous insulin and therefore, would not influence directly the daily insulin requirements of diabetic individuals.

Effects of Comparative Perfusions of Equimolar, Single Component Insulin and Proinsulin in the Human Forearm

Abstract: The human forearm was used as a model system for testing the biological activity of proinsulin (PI) upon peripheral adipose tissue and muscle. Contrasts were made with equimolar perfusions of single component insulin (SCI) in a mean venous perfusion concentration of 263 μU per milliliter and also with SCI perfusions giving venous concentrations of 83 or 47.7 μU permilliliter. Mean venous concentration of PI when perfused alone was equivalent on a molar basis to SCI at 281 μU. per milliliter. PI, at a molar excess of 26:1 (PI:endogenous insulin), shut off lipolysis and promoted potassium uptake but was only slightly effective in promoting glucose uptake. PI was quantitatively similar in effect to 47.7 or 83 μU. per milliliter of insulin upon K flux and blockade of lipolysis but less effective than 47.7 μU. per milliliter in promotion of glucose uptake. When PI and SCI were simultaneously perfused in a molar ratio of 1:1, PI neither augmented nor inhibited SCI actions.

Substrate Studies for Insulin-Specific Protease

Abstract: Insulin-specific protease, a soluble cellular enzyme from rat skeletal muscle which has been purified recently as a single enzyme, has been studied in regard to its substrate specificity, using various immunoreactive and biologically active insulin and proinsulin intermediates. The rate of degradation of pork insulin taken as 100% was compared to other insulin and proinsulin derivatives. Porcine proinsulin intermediates consisting of cleaved proinsulin, desdipeptide, desnonapeptide and destridecapeptide-proinsulin, as well as desalanine, monoarginine and diarginine-insulin, were degraded at 19.8, 25.6, 63.5, 73.7, 101.5, 98 and 98% of the activity of insulin, respectively. Rates of degradation of beef proinsulin, and intermediates I and II were 6, 20.8 and 5.9% of that of insulin with insulin-specific protease, respectively. Studies of Km and V determinations of pork insulin and proinsulin and their intermediates revealed that all the substrates had similar V values (1.0 pmol/min); whereas the Km values (nM) were as follows: insulin, 22.2; desalanine-insulin, 15.8; monoarginine-insulin, 24.4; diarginine-insulin, 24.4; proinsulin, 857.2; cleaved pro-insulin, 234.2; desdipeptide-proinsulin, 176.0; desnonapeptide-proinsulin, 55; and destridecapep-tideproinsulin, 44. Reduced proinsulin, labeled with iodo[14C]acetamide, did not show increased degradability by insulin-specific protease as compared to native proinsulin. These studies suggest that one requirement for optimal substrate activity may be the deblocking of the amino end of the A chain of insulin. The blocking of the amino end, which is present in proinsulin or proinsulin intermediate, will reduce degradability of these substrates by insulin-specific protease. The fact that the cleavage of disulfide bonds of proinsulin resulted in no further activation of proinsulin supports the above and indicates that steric hindrance may have a lesser role in the proinsulin molecule's inability to be degraded by insulin-specific protease.

Proinsulin and Insulin Release with a Human Insulinoma and Adjacent Nonadenomatous Pancreas

Abstract: The availability of surgically obtained samples of an insulin secreting human islet cell adenoma and adjacent nonadenomatous pancreas permitted studies in vitro of biosynthesis and release of proinsulin and insulin moieties. Acid-alcohol extracts of plasma, tumor and normal pancreas were subjected to gel nitration on Sephadex G-50 columns. IRI (immunoreactive insulin and proinsulin) were measured. Proinsulin in tumor represented 40% of the total IRI, in serum 37%, while in normal pancreas, it was only 5%. Studies of unlabelled proinsulin and insulin release revealed that normal human pancreas responded in a manner similar to that reported for normal pancreas of the rat. In the absence of glucose, there was little insulin release. In the presence of glucose, insulin release increased and diazoxide and epinephrine inhibited this glucose effect. No proinsulin release could be found at any time. In contrast, the tumor released proinsulin rapidly even in the absence of glucose, while insulin release was nil. G...

Insulin biosynthesis in the bullhead, Ictalurus nebulosus, and the effect of temperature

Abstract: Insulin biosynthesis in the brown bullhead, Ictalurus nebulosus (Le Sueur), was studied by measuring the incorporation in vitro of [3H]leucine into proteins of the principal islet. The tissue was incubated for 6–15h in Krebs–Ringer bicarbonate buffer with [3H]leucine, supplemented with amino acids and glucose. Proteins, precipitated with trichloroacetic acid and extracted with acid ethanol, were separated by gel-filtration on Biogel P-30 in 3m-acetic acid. Three major components were found after incubation of the islets at 22°C. On the basis of the results of sulphitolysis, biological activity and the demonstrated precursor–product relationship, components I and II were identified as proinsulin and insulin respectively. The third component was not identified. At 12°C, [3H]leucine was incorporated only into proinsulin. No radioactivity was found in insulin or the unidentified component III at 12°C as was found after incubation at 22°C. When the temperature was lowered from 22° to 12°C after 3h of a 15h incubation, decreased conversion of proinsulin into insulin resulted at the lower temperature compared with the control tissue maintained at 22°C. When the temperature was raised from 12° to 22°C at 3h of a 15h incubation, conversion of proinsulin into insulin occurred. No conversion occurred in the control tissue with the temperature maintained at 12°C. No qualitative difference in the incorporation of [3H]leucine into proinsulin and its conversion into insulin at 12° and 22°C could be demonstrated between islet tissue from fish acclimated to less than 12°C or to 22°C. The results suggest that the enzyme(s) responsible for converting proinsulin into insulin in the bullhead may be temperature sensitive with low activity at 12°C.

Human pancreatic islet tumor cells maintained in vitro

Abstract: Tumor cells from two human insulinomas were maintained in culture for more than 6 months. The cultured cells synthesized and secreted proinsulin and insulin; and responded to cyclic AMP (1 mM) and tolbutamide (1 mM) stimulation with increased insulin release. In culture, these cells did not respond to stimulation by high concentrations of glucose (300 mg%) or by glucagon (10 μg/ml).

The glomerular basement membrane of the rabbit kidney on long-term treatment with heterologous insulin preparations of different purity.

Abstract: A total of 90 rabbits were treated with monocomponent insulin (MC Actrapid Novo) and a+b component (proinsulin, intermediates, “dimer”, high-molecular proteins) and their solvents for 3 months without Freund's adjuvant. The insulin-binding capacity of the serum, as an expression of the antibody titre, as well as the light and electron microscopic structure of the glomerular basement membrane were studied. It was shown that on treatment with MC insulin there was no significant antibody formation and no increased occurrence of subepithelial, hump-like, basement membrane protuberances. In contrast, following immunization with a+b component there was a significantly higher antibody titre and a pronounced increase in nodular basement membrane changes of the glomeruli. The findings show that the impurities (a+b component) in the insulins in normal commercial usage are evidently responsible for the higher antigenicity. Further, it can be demonstrated that investigations of glomerular structures allow one to draw conclusions as to the degree of the antigenicity of different insulin preparations although the actual cause of the increased occurrence of basement membrane changes with increasing antigenicity of the insulin preparation is still unclarified. Possible causes which ought to be discussed are deposition of immune complexes and/or increased and structurally atypical basement membrane synthesis.

Hypophysis and function of pancreatic islets. II. The effect of substitution with growth hormone and corticotrophin on insulin secretion and biosynthesis of proinsulin and insulin in isolated pancreatic islets of hypophysectomized rats.

Abstract: Hypophysectomized rats were substituted with varying doses of human or porcine growth hormone (GH) as well as with ACTH for 6 or 12 days. Hypophysectomy was performed in animals of 80 or 170 g body weight either 12 or 4 weeks prior to the onset of the therapy. Increase in weight and the width of the epiphyseal cartilage were determined, insulin secretion and biosynthesis of proinsulin and insulin, were investigated in isolated pancreatic islets of the animals. — No differences were found between the effects of human and poreine GH preparations. Weight gain was similar in rats which had been hypophysectomized at a weight of 80 g either 12 or 4 weeks prior to the substitution. Secretion and biosynthesis of insulin which were both found to be reduced in isolated islets of untreated, hypophysectomized rats, were improved or normalized after substitution with GH (1 mg/kg/day) for 12 days. On the other hand, a therapy with GH for 6 days, even in tenfold daily dose (10 mg/kg), was ineffective in all rats which had been hypophysectomized at a weight of 80 g. Normalisation of lowered levels of blood sugar was the only positive effect observed after an administration of ACTH for 6 or 12 days. — It appears from our findings that, in contrast to the administration of ACTH, GH given to hypophysectomized rats for a longer period in relatively low doses may normalize both reduced secretion and biosynthesis of insulin.