Showing papers on "Proinsulin published in 1969"

Secretion of Proinsulin C-Peptide by Pancreatic β Cells and its Circulation in Blood

Abstract: BIOSYNTHETIC studies with human islet cell tumours1,2, rat2,3 and codfish4 islets, and foetal calf pancreas5 have shown that insulin is formed from proinsulin, a single-chain polypeptide precursor. This protein has subsequently been purified from commercial preparations of crystalline insulin6, and the amino-acid sequences of porcine7 and bovine8 proinsulin have been determined. The molecule begins at the N-terminus with the B-chain sequence of insulin, terminates with the insulin A-chain and bears a connecting segment of thirty (bovine) or thirty-three (porcine) amino-acids linking the two chains. Although 30 per cent of the amino-acid sequences of these connecting segments differ, identical pairs of basic amino-acids occur at each end in both species. Limited tryptic digestion in each case liberates desalanyl–insulin, alanyl–arginine, free arginine and the remainder of the connecting segment as an intact peptide6,7.

Insulin biosynthesis in the rat: demonstration of two proinsulins

Abstract: Proinsulin, a single-chain precursor of insulin, has been detected in biosynthetic studies in vitro,1-4 and in crystalline preparations of insulin from several species.1, 5-7 It appears that proinsulin normally serves neither as a major storage nor secretory form of the hormone.8 Its primary functions seems to be to facilitate the efficient formation of the disulfide bonds of insulin.9 Although several species of fish have two insulins differing slightly in primary structure,10-12 the rat is the only mammal where this is known to be the case.13, 14 As the two rat insulins are nonallelic14 and presumably coded by two distinct genes, a separate proinsulin would be expected to precede each insulin. We have demonstrated the biosynthesis of both insulins15 and of a proinsulin corresponding to each in isolated islets of Langerhans, and have detected probable intermediates in the conversion of these proinsulins to insulin. Labeled peptide material corresponding to the free connecting segment of proinsulin (C-peptide) was detected in insulin-containing fractions isolated from incubated rat islets. It was noted that islets incubated in vitro secreted small amounts of newly synthesized proinsulin as well as the two insulins and essentially equivalent amounts of C-peptide. Increasing the glucose content of the medium selectively enhanced the incorporation of radioactivity into proinsulin and insulin and also seemed to increase the relative proportion of labeled proinsulin appearing in the medium.

Insulin tolerance test in depression.

Abstract: A RECENT investigation from this laboratory 1 demonstrated that psychotic depression is associated with low rates of glucose utilization (k) and high levels of serum insulin following glucose injection. Patients with neurotic depression did not demonstrate these abnormalities. This abnormality, a resistance to endogenous insulin, returned toward normal when the patient improved. These findings, which could not be accounted for by initial levels of serum human growth hormone (HGH), age, or nutritional state, suggested that psychotic depression is associated with a type of endogenous insulin resistance unlike that of any previously described condition. Recent studies by Steiner et al 2 and Shaw and Chance 3 have shown that the routine serum insulin radioimmunoassay measures both insulin and the insulin precursor, proinsulin, but that proinsulin is not biologically active until enzymatically degraded to insulin. It is, therefore, possible that the higher serum

Proinsulin and the Biosynthesis of Insulin

Abstract: THE protein hormone insulin seems to be of perennial interest to biochemists It was among the first to be crystallized and was the first protein whose primary structure was elucidated by Sanger and his collaborators in the 1950's (Fig 1) It is also the first protein that was totally synthesized chemically, this feat having been accomplished in 1964 in several laboratories throughout the world Much interest has centered around the mechanism of biosynthesis of its two-chain structure Two major possibilities exist: the two polypeptide chains might be made on separate ribosomal units and then combined through the formation of disulfide

Plasma insulin: fluctuations in the “big” insulin component in man after glucose and other stimuli

Abstract: Plasma was filtered on G50 Sephadex, and two components of circulating insulin, “little” insulin and “big” insulin, were measured by immunoassay. The former component is indistinguishable from insulin, whereas the latter more closely resembles proinsulin and the other insulin-like substances isolated from the pancreas. In thin normal subjects, the fraction of plasma insulin that was big insulin (per cent “big”). 15-30 min after oral glucose, was less than 5%; per cent big rose 2- to 8-fold over the next hour and by 90-120 min represented 5-29% of the plasma insulin. In young thin subjects with idiopathic glucose intolerance associated with normal concentrations of plasma insulin, an identical pattern of big insulin was observed. In thin subjects in whom elevations of per cent big at 90-120 min during the standard test were only modest, starvation for 48 hr before the glucose administration resulted in a more pronounced rise in the per cent big insulin. Early after glucose administration to obese and acromegalic subjects, the per cent big was higher than in thin subjects. The magnitude of the elevation was roughly correlated with elevations in the fasting plasma insulin concentration. By 90-120 min, the per cent big in obese and acromegalic patients was the same range as in the thin subjects. Intravenously administered arginine and tolbutamide in a small number of subjects yielded a response that was similar to oral glucose; the per cent big was low early after stimulation and increased with time. In two patients with islet cell carcinomas, the per cent big was in the same range as in normal subjects.

Significance of human plasma insulin Sephadex fractions.

Abstract: Fifty-nine samples taken from lean and obese healthy subjects and from patients with diabetes mellitus, myotonic dystrophy, and islet cell adenoma after the administration of oral or intravenous glucose or other insulin secretogogues were fractionated on Sephadex G-50 columns. Eluates corresponding to zones of elutions of porcine pro insulin (“big insulin”) and Regular insulin were assayed for insulin by radioimmunoassay using crystalline human insulin as standard and porcine insulin-I-125 as tracer. In four samples from one patient with islet cell adenoma the big insulin fraction composed 24 to 55 per cent of the apparent insulinimmunoreactivity but in no other case did big insulin exceed 20 per cent of the total and in fifty-one of the fifty-five samples other than from the patient with insulinoma, big insulin contributed less than 15 per cent to the total apparent immunoreactive plasma insulin.

Circulating Insulins "Big" and "Little"

Abstract: Plasma insulin has been characterized by its biologic, immunologic, physical, and chemical similarity to pancreatic insulin. A higher molecular weight heterogenous component (peak "b") of pancreatic insulin that contains the biosynthetic precursor of insulin (proinsulin) has recently been described. Plasma insulin as measured by radioimmunoassay is composed of at least two components. Filtration of plasma on G-50 Sephadex yields a major component ("little" insulin) which is indistinguishable from the major component of pancreatic insulin and a second component "big" insulin which is less retarded by the gel similar to peak "b." Both components react strongly with insulin antisera and are secreted by the pancreas in response to stimuli of insulin secretion. The characteristics of these recently described components of pancreatic and of plasma insulin are described and discussed.

Insulin biosynthesis: effects of carbohydrates and related compounds.

Abstract: Incorporation of tritium-labeled leucine into proinsulin and insulin was used to indicate proinsulin synthesis and insulin formation. In these experiments the incorporation of labeled leucine into proinsulin and that into insulin were closely related. This incorporation was stimulated by addition of glucose and mannose but not by fructose. The glucose- and mannose-stimulated incorporation was inhibited by mannoheptulose. Inhibition by mannoheptulose was not relieved by addition of pyruvate. Pyruvate, with the addition of small amounts of fumarate and glutamate, failed to stimulate proinsulin and insulin synthesis even though insulin secretion was stimulated by tolbutamide or ribose. It was concluded that neither free glucose, nor pyruvate derived from glucose, acts as a signal to initiate biosynthesis. Possibly some more immediate metabolite of glucose or some enzyme or cofactor associated with its metabolism may be involved. Ribose and xylitol failed to stimulate the biosynthesis of proinsulin, and insul...

Immunological properties of bovine proinsulin and related fractions.

Abstract: The immunological interrelationships of bovine proinsulin, its related fractions and insulin are described. Proinsulin has been iodinated and the labeled material purified and characterized. Proinsulin and its intermediate fractions, which could be converted to desalanyl insulin by trypsin, reacted more strongly than insulin or the “nonconvertible” insulin-like material with antiserum to purified proinsulin. The order of reactivity was reversed in favor of insulin when an insulin antiserum was used. Preincubation of the antiserum to purified proinsulin with large quantities of insulin allowed the preferential measurement of bovine proinsulin, and the assay was unaffected by further addition of insulin. The isolated C-peptide from proinsulin was effective in displacing I-131-proinsulin from this antibody. Human and porcine proinsulin reacted weakly with antisera to bovine proinsulin. The immunological differences between proinsulins are far greater than among the corresponding insulins, and imply that the C-peptide of proinsulin contains major antigenic determinants. It is probable that specific antisera will be needed for the optimal assay of each species of proinsulin.

A specific anti-proinsulin serum and the presence of proinsulin in calf serum

Abstract: A specific antibody against proinsulin has been obtained by adsorbing the original anti-proinsulin guinea pig serum with a solid immunosorbent of Sephadex-insulin. The specificity of the antibody against antigenic determinants of proinsulin was established by radioimmunoassay and by passive cutaneous anaphylaxis (PCA) tests. The presence of proinsulin in calf serum has been demonstrated by (a) gel filtration of an acid alcohol extract of the serum followed by immunoassays of the fractions using anti-insulin or the specific anti-proinsulin serum, and (b) direct assay of calf serum with the specific anti-proinsulin serum.

Proinsulin. Single-chain precursor of insulin.

Abstract: The biosynthesis of insulin occurs in the β-cell via a single-chain protein, proinsulin, which is converted to insulin by cleavage of a peptide that connects the COOH terminus of the B chain to the NH 2 terminus of the A chain. The primary structure of porcine proinsulin is known. Differences in the sequences of the porcine and bovine connecting peptides are reflected by immunologic differences between the respective proinsulins. Proinsulin can be detected in the urine and blood of certain diabetic individuals and may be related to another form of blood insulin, "big" insulin. Although much less effective, proinsulin mimics the actions of insulin on isolated adipose and diaphragm tissues. This is not an intrinsic activity but the result of tissue conversion to a molecule(s) with insulin-like effects.

Biosynthesis of insulin in bovine fetal pancreatic slices: the incorporation of tritiated leucine into a single-chain proinsulin, a double-chain intermediate, and insulin in subcellular fractions.

Abstract: This communication reports the biosynthesis of insulin in the bovine fetal pancreatic slices in vitro. Double-chain proinsulin and insulin were found as major components in the mitochondrial-granule fraction of bovine fetal pancreas. Tritiated leucine was incorporated into a single-chain proinsulin, a double-chain proinsulin, and insulin. Subcellular fractionation of the slices incubated with tritiated leucine showed that radioactive single-chain proinsulin was present in the deoxycholate-soluble microsomal fraction, deoxycholate-insoluble microsomal fraction, and mitochondrial-granule fraction. Labeled double-chain proinsulin and insulin were present in the deoxycholate-soluble microsomal and mitochondrial-granule fractions. These results are consistent with the hypothesis that insulin is synthesized as a single-chain polypeptide on the ribosomes, and that intracellular proteolysis in the subcellular membranous organelles and beta-granules converts the single-chain proinsulin to insulin via a double-chain intermediate.

The effect of proinsulin on the immunoassay of insulin and its possible relation to states of hyperinsulinemia.

Abstract: The measurement of insulin or insulin-like moieties by a variety of bioassay and immunologic procedures has resulted in the designation of such nonspecific varieties as typical and atypical insulin (1), bound and unbound insulin (2), suppressible and nonsuppressible insulin (3). Insulin determined by the radioactive immunoassay and referred to as immunoreactive insulin, IRI, has been judged by most to represent an accurate appraisal of absolute insulin levels in biologic fluids (4); as such, its biologic activity, although inferred, has never been entirely certain. In the immunoassay method, the immunospecificity of antigen to specific antibody is of necessity assumed. Accordingly, if the reaction between insulin-I131, insulin antiserum, and endogenous insulin is specific, then serial dilution of serum containing IRI will yield comparable assay measurements over the range of dilutions studied.In this laboratory where a modification of the Yalow and Berson chromatographic method (4) or the dextran-coated c...

Changes of Immunoreactivity of Proinsulin induced by Rat Tissue Extracts

Abstract: PROINSULIN has little or no biological activity1, but is present in the circulation2 and produces insulin-like effects when injected into normal animals3,4 and alloxan diabetic rats5. It has no effect, however, when injected into eviscerated, hepatectomized rats4.

Experimentelle Untersuchungen über die blutzuckersenkende Wirkung von Rinder-Proinsulin

Abstract: An Ratten und Mausen wird der Blutzucker durch ein aus Rinderinsulin gewonnenes Proinsulin (Schmidt und Arens [6]) gesenkt. Bei gefutterten Ratten und nuchternen Mausen ist das WirkungsVerhaltnis Proinsulin: Insulin = 1∶4, bei nuchternen Ratten 1∶2, wenn aquiznolar dosiert und das Maximum der Blutzuckersenkung gewertet wird. Die Blutzuckersenkung verlauft nach Proinsulin protrahierter als nach Insulin. Proinsulin, welches in vitro mit Trypsin inkubiert wurde, wirkt quantitativ und zeitlich wie Insulin. Bei pankreatektomierten, bei nephrektomierten und bei zweidrittelhepatektomierten Ratten wirkt Proinsulin ebenso wie an Normaltieren. Auch alloxandidbetische Mause sprechen auf Proinsulin wie intakte Mause an. Die hypoglykamische Wirkung des Proinsulins tritt wahrscheinlich deshalb verzogert ein, weil das aktive Insulin in einem zeitabhangigen proteolytischen Prozes erst entstehen mus. Dies konnte auch die Ursache fur eine von Rubenstein et al. [5] zitierte, fehlende Wirkung von Proinsulin im Mausekrampftest sein.

Insights about diabetes and hyperinsulinism gained from the insulin immunoassay

Abstract: In less than a decade the supersensitive insulin immunoassay has crystallized basic aspects of sugar metabolism from a confusion of conflicting concepts into a hard core of integrated knowledge. It has provided ultimate proof, for example, that the supreme insulin-secretory stimulus is hyperglycemia and that sulfonylureas lower blood sugar only by stimulating insulin release. The isolation of the insulin precursor proinsulin is hailed as the most monumental discovery since that of insulin itself.

Blutzuckerwirkimg von Proinsulin / The Effect of Proinsulin on Blood Sugar

Abstract: Steiner et al. [2] isolierten aus tierischem und menschlichem Pankreas ein Proinsulin, welches 29 Aminosauren mehr enthalt als das aus 51 Aminosauren zusammengesetzte Insulin. Durch Proteinasen werden die 29 Aminosauren als einheitliches Bruchstuck abgespalten. Dadurch wird aus der inaktiven Vorstufe das aktive Insulin gebildet. — Da bisher insulinahnliche Wirkungen von Proinsulin an isolierten Fettzellen, am Froschsartorius und am isolierten Rattenzwerchfell nicht gefunden wurden, haben wir mit einem von Schmidt u. Arens [1] aus kauflichem Rinderinsulin gewonnenen Proinsulin gepruft, ob die Hormonvorstufe in vivo, d.h. bei Bedingungen, unter denen sie zu aktivem Insulin umgewandelt werden kann, den Blutzucker herabsetzt.