Showing papers on "Proinsulin published in 1979"

Endocrine pancreas: three-dimensional reconstruction shows two types of islets of langerhans.

Abstract: Three-dimensional reconstructions of islets of Langerhans, based on immunofluorescent staining of successive serial sections with antiserums to insulin, glucagon, somatostatin, and pancreatic polypeptide reveal a marked difference in the number of cells containing glucagon and pancreatic polypeptide depending on the anatomical location of the islet in the pancreas. The two types of islets are situated in regions of exocrine tissue that are drained by different excretory ducts. This demonstration contradicts the assumption that all islets in the pancreas are similar in their endocrine cell content.

Effects and binding of insulin-like growth factor I in the isolated soleus muscle of lean and obese mice: comparison with insulin.

Abstract: The effects and binding of insulin-like growth factor-I (IGF-I) in skeletal muscle were investigated in the isolated mouse soleus muscle from normal lean and goldthioglucose- obese mice. In muscles from lean mice, IGF-I stimulated 2-deoxyglucose uptake, glycolysis, and glycogen synthesis and activated glycogen synthase. The latter effect occurred in the absence of glucose in the incubation medium, as was also observed with insulin. The maximal effects of IGF-I and insulin on 2-deoxyglucose uptake were not additive. IGF-I was about 4–9% as potent as insulin (half-maximal effects occurred with 5-14 nM IGF-I) and nearly as effective as insulin. A specific binding of [125I]iodo-IGF-I was observed, which was not inhibited by unlabeled insulin or proinsulin. IGF-I was less than 1% (on a molar basis) as potent as insulin in competing with [125I]iodoinsulin binding. Unlike [125I]iodoinsulin binding, [125I]iodo-IGF-l binding was similar in muscles from lean and goldthioglucose-obese mice. As observed with insulin,...

Identification and processing of proglucagon in pancreatic islets

Abstract: Immunoprecipitation and tryptic peptide analysis of newly synthesized proteins from rat islets have identified an 18,000 molecular weight (MW) protein as proglucagon. Conversion of this precursor was kinetically similar to the conversion of proinsulin and resulted in the formation of both pancreatic glucagon and a 10,000-MW protein lacking this hormonal sequence.

Nucleotide sequence of a cDNA clone encoding human preproinsulin

Abstract: Insulin consists of two polypeptide chains, A (21 amino acids) and B (30 amino acids), linked by disulphide bonds. Both chains are derived from one precursor, proinsulin, which includes a connecting peptide (C) between the A and B chains, and which is excised before the secretion of insulin from the pancreatic B cells1. The observation that the C-peptide varies between species, in contrast to the highly conserved A and B sequences1–3, is consistent with the theory that it serves a purely structural function in insulin synthesis. During in vitro translation of insulin mRNA, a larger peptide containing about 25 additional residues at the N-terminal end (preproinsulin) is the primary product4–7. The prepeptide is cleaved to leave proinsulin during transport into the endoplasmic reticulum and is thought to direct this process specifically8. Using automated amino acid sequence analysis, the partial amino acid sequences of the prepeptide regions of bovine, rat, sea raven and anglerfish preproinsulin have been established4,5,7. The nucleotide sequences of the cloned cDNA and gene coding for rat insulin I have confirmed the amino acid sequence of rat proinsulin I, and have also predicted the sequence of the prepeptide9–11. Like the prepeptides of other secreted proteins, this prepeptide has a prominent hydrophobic region12. We report here the cloning of a cDNA prepared from human insulin mRNA and an analysis of the nucleotide sequence of the cloned molecule including the region coding for the prepeptide and portions of the 5′- and the 3′-untranslated regions of the molecule. We also compare the structure of the human molecule with the previously reported rat mRNA9–11.

Characterization of circulating insulin and proinsulin-binding antibodies in autoimmune hypoglycemia.

Abstract: Five patients with fasting and(or) postprandial hypoglycemia were found to have insulin antibodies in the absence of previously documented immunization. Studies on the equilibrium-binding of insulin to the autoantibodies revealed two classes of binding sites with association constants and binding capacities analogous to those of insulin antibodies from insulin-treated diabetic patients. Similarly, no consistent differences in these parameters were found in both groups of patients with insulins of bovine, porcine, and human origin. Proinsulin (C-segment directed) antibodies capable of binding bovine or porcine proinsulin were present in 10 of 10 and 9 of 10 insulin-treated diabetics serving as controls, respectively, and, when present, provide incontrovertible evidence of exogenous insulin administration. No such antibodies could be detected in the hypoglycemic patients with autoimmune insulin antibodies. The kinetics of dissociation of the insulin-antibody complexes were consistent with the existence of two classes of antibody sites. The corresponding dissociation rate constants were large enough to predict that significant amounts of free hormone may be generated by this mechanism and provide a plausible pathogenesis for the hypoglycemia in these patients.

Purification and characterization of proinsulin mRNA from rat B-cell tumor.

Abstract: Proinsulin mRNA was purified from rat B-cell tumor induced by streptozotocin and nicotinamide. The purification was achieved through the use of oligo(dT)-cellulose chromatography and sucrose gradient centrifugation. As judged by translational activity in a cell-free protein-synthesizing system of wheat germ, proinsulin mRNA was purified 362-fold from total nucleic acid of the B-cell tumor. Purified proinsulin mRNA migrated as a single symmetrical peak on sucrose gradient centrifugation and as a single band on polyacrylamide gel electrophoresis in the presence of formamide. The translation product of purified proinsulin mRNA in the cell-free protein-synthesizing system was found to be only preproinsulin, as analyzed by dodecylsulfate/polyacrylamide gel electrophoresis and immunoprecipitation. The molecular weight of proinsulin mRNA, determined by polyacrylamide gel electrophoresis in the presence of formamide, was approximately 200000 (equivalent to approximately 570 nucleotides). Almost all of the proinsulin mRNA was bound to oligo(dT)-cellulose, and the translation of proinsulin mRNA in a cell-free protein-synthesizing system of wheat germ was completely inhibited by 7-methylguanosine 5′-phosphate. These results indicated that the nucleotide sequences in proinsulin mRNA consisted of 58% translatable sequences and 42 % untranslatable sequences, presumably containing a capped structure, a 7-methylguanosine residue, at the 5′ terminus and a poly(A) sequence at the 3′ terminus. The translation product of purified proinsulin mRNA, preproinsulin, was found to consist of two types of preproinsulin I (57%) and preproinsulin II (43%). This result indicated that the isolated proinsulin mRNA consisted of two types of proinsulin I mRNA and proinsulin II mRNA in approximately equal amounts. Complementary DNA was synthesized with proinsulin mRNA as a template. From the experiment of hybridization kinetics under conditions of excess RNA, the r0t1/2, of proinsulin mRNA was found to be 2.1 × 10−4 mol 1−1 s. This result also confirmed that proinsulin mRNA had been isolated in a high degree of purity.

Identification of Human Glandular Kallikrein in the Beta Cell of the Pancreas

Abstract: To determine the cellular localization of glandular kallikrein in the human pancreas, immunohistochemical studies were performed with a mono-specific antibody against the antigenically identical urinary kallikrein (urokallikrein). The localization of glandular pancreatic kallikrein to the beta cells of the islets was the same as that of insulin in normal human pancreas and in two islet-cell tumors. When beta cells were lacking in islet-cell tumors or in the pancreas of a patient with juvenile-onset diabetes, kallikrein antigen was not detectable. Anti-urokallikrein absorbed with purified urinary or pancreatic kallikrein no longer identified a pancreatic antigen, whereas absorption with insulin had no effect. The beta-cell localization of human pancreatic kallikrein, an endopeptidase that, in concert with carboxypeptidase B, converts bovine proinsulin to a polypeptide with the electrophoretic mobility of insulin, suggests that pancreatic kallikrein may be involved in the physiologic activation of ...

Evidence for the Existence of a Biosynthetic Precursor for Somatostatin

Abstract: The purpose of this study was to determine if the somatostatin in pancreatic islets is synthesized via a precursor-product pathway. Anglerfish islets were subjected to pulse-chase incubations with 3 H-tryptophan 14 C-isoleucine. Islet extracts were subjected to gel filtration on Bio-Gel P-10. During chase incubations in the presence of cycloheximide, a linear increase of 3 H-radioactivity in the somatostatin-containing portion of the eluate was observed while 14 C-label did not accumulate in this region. A concomitant diminution of both 3 H- and 14 C- radioactivity in the 8-15-kilodalton region of the eluate occurred during the chase periods, suggesting a transfer of label from larger to smaller peptides. The labeled peptides found in the 8-15-kilodalton range were subjected to treatment with 8 M urea, 8 M urea−5% thioglycolic acid, 6 M guanidine hydrochloride, and 8 M guanidine hydrochloride−5% thioglycol. No appreciable loss of radioactivity was apparent after any of these treatments. Incubation of these same peptides with cell-free supernate from unlabeled islet tissue or with trypsin yielded a tryptophan-labeled peptide having the molecular size, electrophoretic mobility, and immunoreactivity of anglerfish and synthetic somatostatin. These results suggest that a precursor peptide having a molecular size near that of proinsulin is used in the biosynthesis of somatostatin.

Demonstration and partial characterization of insulin receptors in human platelets.

Abstract: Recently, evidence has been reported to suggest that human platelets like several other circulating blood cells may bind insulin. To examine whether human platelets contain specific insulin receptors, washed human platelets suspended in Hepes buffer were incubated at 24 degrees C with 125I-insulin in the presence and absence of unlabeled insulin and specific insulin binding was determined. Insulin binding by platelets increased progressively with time of incubation to reach a maximum at 3 h and was proportional to the number of platelets in the incubation mixture. Maximum insulin binding was observed at pH 8. Insulin degradation by platelets as assessed by TCA precipitability and reincubation studies was minimal. Scatchard analysis of the binding data and dissociation studies revealed evidence of negative cooperativity of the platelet insulin receptor. A high affinity dissociation constant of approximately equal to 3 X 10(9) M-1 was determined and the concentration of platelet insulin receptors was estimated as 25 binding sites/micron2 platelet surface area. Binding of 125I-insulin by platelets was inhibited by unlabeled porcine insulin and to a lesser extent by catfish insulin and porcine proinsulin but not by glucagon, prolactin, growth hormone, and thrombin. The findings indicate that human platelets contain specific insulin receptors. The significance of the platelet insulin receptor, particularly with respect to altered platelet function in diabetes mellitus, remains to be determined.

The Insulin Receptor and Insulin of the Atlantic Hagfish: Extraordinary Conservation of Binding Specificity and Negative Cooperativity in the Most Primitive Vertebrate

Abstract: The North Atlantic hagfish, a cyclostome that is representative of the most primitive vertebrates still alive, diverged from the other vertebrates about 500 million years ago. Hagfish insulin, which differs from porcine insulin in 18 (38%) of its amino acids, had a potency of 5-10% that of porcine insulin in stimulating glucose oxidation and deoxyglucose transport in rat adipocytes and was 5-10% as potent as porcine insulin in binding to insulin receptors on rat adipocytes and human (IM-9) lymphocytes. Like all other naturally occurring insulins, hagfish insulin accelerated the dissociation of 125 I-porcine insulin from insulin receptors and the degree of the acceleration was related to its occupancy of the receptor. The insulin receptor of the hagfish erythrocyte showed the time, temperature, and pH dependence of binding and the negative cooperativity that are characteristic of all other insulin receptors. That the negative cooperativity is fully conserved in such an ancient insulin and receptor suggests that it is an important functional feature of this hormone-receptor system. The hagfish receptor showed the same absolute affinity and rank order of preference for insulins and insulin analogues (chicken > pork > proinsulin > guinea pig > desoctapeptide) found with other receptors of less primitive vertebrates, which supports the conclusion that the receptor for insulin is functionally better conserved evolutionary than the hormone. However, uniquely, hagfish insulin was more potent in binding to hagfish receptors than to mammalianreceptors; with all other species of insulins studied, the affinity of the hormone for homologous receptor was the same as for receptors of heterologous species.

Sequential cleavage of proinsulin by human pancreatic kallikrein and a human pancreatic kininase.

Abstract: A pancreatic endopeptidase localized to the β-cells of the pancreas by immunohistochemical techniques has been purified to homogeneity by following its functional and antigenic characteristics as a glandular kallikrein (EC 3.4.21.8). The enzyme gave a single stained band on alkaline disc gel electrophoresis which corresponded in location with the kinin-generating activity eluted from a replicate gel, was of 54,000 molecular weight by gel filtration, was devoid of caseinolytic activity, elicited a monospecific antiserum in a rabbit, and gave a line of complete identity with a single constituent in pancreatic extract, crude urine, and purified urokallikrein when analyzed with monospecific antibody to urokallikrein. The pancreatic glandular kallikrein generated three cleavage products of increasing anodal mobility from bovine and porcine proinsulin, and the presence of pancreatic kininase or bovine carboxypeptidase B increased the quantity of these products. Although the conversion products did not correspond to diarginyl- and monoarginylinsulin, the product of intermediate mobility was also obtained when proinsulin was treated with a low concentration of trypsin in the presence of kininase. The most rapidly migrating product did correspond to desalanylinsulin in the reference standard. Kininase alone had no action on proinsulin, and aprotinin prevented cleavage by kallikrein alone or in combination with kininase. Although the chemical structure of the proinsulin cleavage products has not been established, human pancreatic kallikrein is considered a putative activator of proinsulin because of its location in the β-cell, its preferential action on proinsulin and kininogen as compared to azocasein, and its capacity to generate insulin intermediate products that are further modified by human pancreatic kininase or bovine carboxypeptidase B.

Role of Microtubules in the Synthesis, Conversion, and Release of (Pro)Insulin

Abstract: A B S T R A C T In the pancreatic B cell, microtubules are thought to be involved in the process of insulin release. Their possible participation in the sequence of events leading from the biosynthesis and conversion of proinsulin to the release of newly synthesized insulin was investigated in rat isolated islets exposed to colchicine (0.1 mM). When the islets were preincubated for 30 min with colchicine and [3H]leucine and, thereafter, incubated for two successive periods of 90 min each, still in the presence of colchicine, the release of preformed insulin was progressively inhibited and that ofnewly synthesized hormone delayed. When the islets were preincubated for 120 min with colchicine, subsequently pulse-labeled with [3H]leucine, and eventually examined by ultrastructural autoradiography, the export of newly synthesized proinsulin out of the rough endoplasmic reticulum, its transit through the Golgi complex, and its eventual packaging in secretory granules were all retarded. This situation was associated with a delayed conversion of proinsulin to insulin. Under the same experimental conditions, colchicine failed to affect the oxidation of glucose and adenylate charge in the islets. The effect of colchicine upon the release ofpreformed and newly synthesized insulin was not reproduced by lumicolchicine. It is concluded that colchicine interferes with the system controlling the intracellular transfer ofsecretory material from site of synthesis to site of release. This interference is likely to be linked to the effect of colchicine on microtubules.

Role of microtubules in the synthesis, conversion, and release of (pro)insulin. A biochemical and radioautographic study in rat islets.

Abstract: In the pancreatic B cell, microtubules are thought to be involved in the process of insulin release. Their possible participation in the sequence of events leading from the biosynthesis and conversion of proinsulin to the release of newly synthesized insulin was investigated in rat isolated islets exposed to colchicine (0.1 mM). When the islets were preincubated for 30 min with colchicine and [3H]-leucine and, thereafter, incubated for two successive periods of 90 min each, still in the presence of colchicine, the release of preformed insulin was progressively inhibited and that of newly synthesized hormone delayed. When the islets were preincubated for 120 min with colchicine, subsequently pulse-labeled with [3H]leucine, and eventually examined by ultrastructural autoradiography, the export of newly synthesized proinsulin out of the rough endoplasmic reticulum, its transit through the Golgi complex, and its eventual packaging in secretory granules were all retarded. This situation was associated with a delayed conversion of proinsulin to insulin. Under the same experimental conditions, colchicine failed to affect the oxidation of glucose and adenylate charge in the islets. The effect of colchicine upon the release of preformed and newly synthesized insulin was not reproduced by lumicolchicine. It is concluded that colchicine interferes with the system controlling the intracellular transfer of secretory material from site of synthesis to site of release. This interference is likely to be linked to the effect of colchicine on microtubules.

Intermediary metabolism and insulin secretion from isolated rat islets of Langerhans.

Abstract: (Ail are verbatim summaries) Grodsky, G. M.; Batts, A. A.; Bennett, L. L; Vcella, C ; McWilliams, N. B.; and Smith, D. F. (Metab. Res. Unit and Depts. of Biochem. and Physiol., Univ. of California Sch. of Med., San Francisco): Effects of Carbohydrates on Secretion of Insulin from Isolated Rat Pancreas. Am. J. Physiol. 205:638, 1963. The effect of carbohydrates on the secretion of immunochemically measurable insulin was studied in an isolated perfused pancreatic preparation from the rat. Degradation of circulating insulin (as measured by chromatographic examination of added insulin-l) was less than 15% during the 4-hr experimental period. Without the addition of glucose, or at glucose concentrations of less than 50 mg/100 ml, insulin secretion was not detectable. At glucose concentrations of 50-150 mg/100 ml, insulin secretion occurred immediately and persisted throughout the experimental period. Insulin secretion was further increased by increasing glucose concentration to 150-500 mg/100 ml. The incidence of islet cell degranulation increased with increasing insulin secretion, suggesting that glucose stimulated secretion of stored insulin faster than synthesis of insulin de novo. Galactose, xylose, L-arabinose, pyruvate, and 2-deoxyglucose in concentrations of 600 mg/100 ml did not stimulate insulin secretion. Mannose stimulated the pancreas equally as well as glucose. Fructose was also active, but was less effective than glucose. Neither 2-deoxyglucose nor galactose blocked the insulin secretion by glucose. The data suggest that secretion of insulin is stimulated by a metabolite or a product resulting from the metabolism of glucose which can also be supplied by other metabolizable sugars. Coore, H. G.; and Randle, P. J. (Dept. of Biochem., Univ. of Cambridge): Regulation of Insulin Secretion Studied with Pieces of Rabbit Pancreas Incubated in Vitro. Biochem. J. 93:66, 1964.1. The effect of various factors on the rate of release of insulin from pieces of rabbit pancreas incubated in vitro has been studied by estimating the insulin concentration in the medium after incubation by immunological assay with insulin-antibody precipitate, and the conditions necessary for eliciting consistent responses have been defined. 2. Insulin release was accelerated by D-glucose at concentrations above 0.35-0.7 mg./ml. and by D-mannose (3 or 6 mg./ml.), but not by D-galactose, 3-O-methyl-D-glucose, D-fructose, D-ribose or sodium D-gluconate (3 mg./ ml.), D-2-deoxyglucose (3 or 6 mg./ml.), A/-acetyl-D-glucosamine (15 mg./ml.) or D-mannoheptulose (3 mg./ml.). The stimulating effect of glucose (3 mg./ml.) on insulin release was abolished by mannoheptulose but not by 2-deoxyglucose, 3-O-methylglucose, ribose or A/-acetylglucosamine. 3. Insulin release at a low glucose concentration (0.6 mg./ml.) was accelerated by tolbutamide (200 /ug./ml.) but not by anoxia, 2,4-dinitrophenol, salicylate, p-phenylenediamine or phenazine methosulphate. The rate of insulin release at a high glucose concentration (3 mg./ml.) was markedly diminished by anoxia, 2,4-dinitrophenol (250 AIM), salicylate (5 ITIM), p-phenylenediamine (1 mM) and phenazine methosulphate (100 /AM), but not by malonate (10 mM). The stimulating effect of tolbutamide (unlike that of glucose) was not influenced by mannoheptulose. 4. The stimulating effect of glucose (3 mg./ml.) on insulin release was augmented by the presence in the medium of glutamate, fumarate and pyruvate. At a low glucose concentration (0.6 mg./ml.) neither these acids nor octanoate, acetoacetate or /3-hydroxybutyrate influenced the rate of insulin

Familial hyperproinsulinemia: partial characterization of circulating proinsulin-like material.

Abstract: Familial hyperproinsulinemia is an autosomal dominant defect that is associated with strikingly elevated levels of serum proinsulin-like material. Our studies show that trypsin converts familial hyperproinsulinemia proinsulin to insulin more slowly than it converts a 131I-labeled porcine proinsulin marker. Molar yields of insulin indicated that the material may be an intermediate proinsulin. Studies with two human C-peptide antisera that differ in their relative immunoreactivity with human C-peptide and proinsulin showed that the two antisera reacted equally with familial hyperproinsulinemia proinsulin, suggesting that it is a partially cleaved proinsulin intermediate. Sulfitolysis of highly purified material to break the inter- and intra-chain disulfide bridges and subsequent adsorption on a specific B-chain antibody covalently bound to Sepharose beads showed that the C-peptide was still connected to the B-chain. These data indicate that familial hyperproinsulinemia proinsulin is normally cleaved at the C-peptide-A-chain linkage site. A structural abnormality appears to underlie familial hyperproinsulinemia proinsulin, which impairs its cleavage at the B-chain-C-peptide linkage site.

Measurement of human proinsulin by an indirect two-site immunoradiometric assay

Abstract: An indirect two-site immunoradiometric assay is described for the measurement of human proinsulin in plasma. Polyethylene tubes coated with purified guinea-pig antibodies to insulin were used to extract proinsulin and insulin from plasma. Rabbit antibody to human C peptide was then added to react with the C-peptide moiety of the bound proinsulin. The uptake of this antibody was measured by the subsequent binding of 125I-sheep antibody to rabbit IgG. The binding of radioactivity to the tubes was a function of the proinsulin concentration in the sample. The sensitivity of the assay was 0.006 pmol/ml. Only 200 microliters of plasma was required in the assay and the 125I-labelled antibody was produced from readily available reagents. The polyethylene tubes remained stable for at least 5 months after coating. The mean fasting proinsulin level was 0.009 pmol/ml in sixteen normal subjects and 0.025 pmol/ml in twelve maturity onset diabetics. Oral glucose produced an 8 fold increase in proinsulin concentration but a decline in the plasma proinsulin/insulin molar ratio. Four patients with insulinoma had extremely elevated proinsulin levels and proinsulin/insulin ratios.

Stimulation of (pro-)insulin biosynthesis and release by gastric inhibitory polypeptide in isolated islets of rat pancreas

Abstract: (Pro-)Insulin biosynthesis ([3H]leucine incorporation) and insulin secretion were studied in collagenase-isolated rat islets incubated for 3 hours at 1 and 2 mg/ml glucose in the presence of gastric inhibitory polypeptide (GIP). GIP augmented [3H]leucine incorporation and release of insulin at both glucose concentrations. In a second series of experiments it was found that an amino acid mixture was without influence on the insulotrophic action of GIP. Combined stimulation of insulin release by GIP and glucagon did not result in higher insulin output than observed in the presence of each substance alone. Thus GIP, in constrast to many other gastrointestinal peptides, however similar to glucagon, enhances not only release but also biosynthesis of insulin. This insulinotrophic action can be observed already at a glucose concentration of 1 mg/ml. The results underline the outstanding role which GIP appears to play in the regulation of beta-cell function.

Binding of 125I-insulin to the isolated glomeruli of rat kidney.

Abstract: To investigate a possible action of insulin on the glomerulus, the binding 125I-insulin to the isolated glomeruli prepared from rat kidney was examined. When incubated at 22 degrees C, 125I-insulin binding proceeded with time and reached a steady state at 45 min at which time nonspecific binding was less than 25% of total binding. A small fraction of 125I-insulin was degraded during incubation. This binding was specific to insulin in that it was inhibited by unlabeled porcine and beef insulins and to a lesser extent by porcine proinsulin and desalanine-desasparagine insulin, but not by glucagon, parathyroid hormone, vasopressin, calcitonin, and angiotensin II. Increasing concentrations of nonlabeled insulin displaced 125I-insulin binding in a dose-dependent fashion. Scatchard plot of the data was curvilinear consistent with either two classes of receptors with different affinities or a single class of receptors that demonstrate negative cooperativity. The addition of excess nonlabeled insulin to the glomeruli preincubated with 125I-insulin resulted in a rapid dissociation of approximately or equal to 70% of bound 125I-insulin. Insulin decreased the increments in glomerular cyclic AMP levels by epinephrine and by prostaglandin E2, but not those by histamine. These data showed the presence of specific insulin receptors in the glomeruli, and that insulin action may be, at least in part, through modulation of glomerular cyclic AMP concentrations. Such action of insulin may underlie the alteration in glomerular ultrafiltration and the glomerular ultrafiltration and the development of glomerular lesions in diabetes mellitus, a disease in which insulin deficiency or the tissue resistance to insulin exists.

Synthesis and Accumulation of Proinsulin and Insulin during Development of the Embryonic Rat Pancreas

Abstract: Endocrine B cells differentiate normally in embryonic rat pancreatic rudiments cultured in vitro. The specific concentration of immunoreactive insulin based on total protein increases by about 1000-fold during the developmental period, corresponding to days 13–20 of gestation. The rate of (pro)insulin synthesis, measured from the level of radioactive leucine incorporated into insulin, quantitatively accounts for the insulin accumulated during this period. In addition, the relative incorporation of leucine into proinsulin compared to insulin is constant during development and is similar to that found in the B cells of adult islets. Thus, there appears to be no significant change in the rate of conversion of proinsulin to insulin during B cell differentiation.

The role of the cytoskeleton in pancreatic B-cell function.

Abstract: The ultrastructural organization of B-cell microtubular-microfilamentous system, its alteration by several pharmacological agents and the concomitant changes in the dynamics of insulin release, the biochemical characterization of islet tubulin and actin, the involvement of microtubules in the process of proinsulin biosynthesis and conversion, the analysis of motile events in endocrine pancreatic cells, the possible participation of microtubular-microfilamentous structures in cell surface organization of the B-cell, and the anomaly of the microtubular apparatus in certain pathological conditions are reviewed. The experimental data support the view that microtubules and microfilaments play an essential role in the process of insulin synthesis and release, as well as in the mechanism of glucagon secretion.

Insulin receptors in cultured mouse retinal cells.

Abstract: The binding of125I-insulin to uncloned and cloned cultures of mouse retinal cells has been investigated. At 15° C, binding of the hormone reached a steady state by 60 min, while at 37° C equilibrium was reached earlier but at a lower level than at 15° C. Porcine insulin, porcine proinsulin and guinea pig insulin displaced labelled insulin in proportion to their known biological potency. A sharp pH dependence of the hormone binding was observed with an optimum at pH 7.8. The dissociation rate of the125I-insulin was increased in the presence of unlabelled hormone, suggesting the existence of negative cooperativity in the insulin-receptor interaction. The availability of established retinal cell lines with insulin receptors should facilitate the study of the insulin-retina interactions in a controlled in vitro system.

Specificity of radioimmunoassays for relaxin.

Abstract: The specificities of two radioimmunoassays (RIA) for relaxin, based upon crude porcine relaxin (NIH-R-P1; RIA I) and a highly purified porcine relaxin (RIA II) have been studied concurrently using purified hormones and plasma samples. A labelled fraction, selected from radio-iodinated NIH-R-P1 and used in that RIA, was also bound to antiserum raised to the highly purified relaxin. Hence a third RIA was possible in which both the crude and the purified relaxins inhibited in the ng/ml range. Porcine insulin and the connecting peptide of porcine proinsulin did not inhibit any of the assay systems whereas porcine proinsulin did inhibit in each assay at the microgram/ml range. Concurrent measurements by assays I and II have been made in sheep plasma obtained during both delivery of the lamb and suckling. The peak values obtained by assays I and II are 3 and 6 min out of phase during suckling and delivery respectively; the NIH-R-P1 relaxin immunoactivity appearing first. The plasma inhibition curves of both appear to be the sum of individual contributions from relaxin and relaxin-like peptides, such as prorelaxin and its fragments, as seen by different antisera. Both assays, however, give qualitatively similar indices of relaxin immunoactivity. The RIA developed for the more purified peptide would be expected to yield a better quantitative estimate of relaxin secretion but this, like specificity, cannot be shown absolutely.

Precursors in the biosynthesis of insulin and other peptide hormones

Abstract: SUMMARY Recent studies have established that limited intracellular proteolysis may participate at several stages in the biosynthesis of many small polypeptide hormones, as exemplified by insulin Conversion of proinsulin to insulin by trypsin-and carboxypeptidase B-like enzymes occurs in the pancreatic B cell after the peptide has been sequestered from the cytosolic compartment and transported to the Golgi area An earlier role for proteolysis concerns the conversion of the initial translation product, preproinsulin, to proinsulin during its compartmentalization in the rough endoplasmic reticulum Preproinsulin, a methionine-initiated peptide which contains a hydrophobic 24-residue NH 2 -terminal extension, was originally identified as the product of the cell-free translation of insulin mRNA, but more recently it has also been detected in small amounts in intact rat islets Its rapid formation and disappearance during pulse-chase studies are consistent with its role as a biosynthetic precursor and with the proposal that the prepeptide region serves as a leader sequence in the formation of the ribosome-membrane junction and in the vectorial discharge of the peptide A model for segregation is presented, based on important structural features and physical properties of the prepeptides which may enable these to enter and span the microsomal membrane Studies on insulin mRNA obtained from transplantable islet cell tumors indicate that the major component contains approximately 600 nucleotides and possesses a 5′ “cap” structure as well as a 3′ polyadenylate “tail” A closer examination of the insulin tumor mRNA has also revealed two larger polyadenylated RNA fractions having apparent molecular weights of 280,000 and 360,000 These forms, which hybridize to the rat insulin genes and which were shown by ribonuclease T 1 digests to share nucleotide sequences with the major mRNA component, may represent precursors of the mature template These results indicate that several sequential stages of macromolecular processing are required in the formation of the secreted hormone

Reconstitutiom of the Solubilized Insulin Receptor in Phospholipid Vesicles1

Abstract: The insulin receptor was solubilized from turkey erythrocyte membranes by extraction with 1% β-octylglucopyranoside. Insulin binding was enhanced when the solubilized material was reconstituted in phospholipid vesicles. The affinity of the reconstituted vesicles for various insulins was similar to that of the intact membranes: porcine insulin > proinsulin > desoctapeptide insulin. A curvilinear Scatchard plot was obtained for insulin binding to the reconstituted system at 15d`C. A high affinity association constant of 1.4 X 109 M-1 was obtained from the Scatchard plot. This is a four-fold increase over the value for the turkey erythrocyte membrane, which contains more highly saturated phospholipids. This suggests that the insulin receptor may be sensitive to the lipid composition of the membranes in which it is embedded.

Evaluation of a commercial enzyme immunoassay for insulin in human serum, and its clinical application.

Abstract: We applied a "sandwich" method, with use of beads coated with anti-insulin serum and of peroxidase-labeled anti-insulin serum, to an enzyme immunoassay of insulin in human serum. 5-Aminosalicylic acid was used as the substrate for the enzymic reaction. As little as 5 milli-int. units of insulin per liter of serum insulin was detectable. Reproducibility was satisfactory, but extraordinarily high concentrations of proinsulin and of hydrogen donors such as reduced glutathione affect results of the assay. Values determined by our enzyme immunoassay and by double-antibody radioimmunoassay correlated highly (r = 0.938, p less than 0.001, n = 216). We recommend this method for use in the clinical laboratory.

Insulin allergy: reaginic antibodies to insulin and proinsulin.

Abstract: To investigate the involvement of proinsulin (one of the contaminant proteins of therapeutic insulin) in systemic insulin allergy, we studied seven diabetic patients who presented with generalized insulin allergy. Commercial and highly purified (single component) insulins produced almost identical responses on the intradermal tests. In all cases, [125I]bovine insulin and [125I]bovine proinsulin bound to the circulating reaginic immunoglobulins. The binding of [125A]proinsulin was blocked completely by unlabeled insulin and was uneffected by bovine C-peptide, indicating a cross-reaction of proinsulin with antinsulin reaginic immunoglobulins and the absence of proinsulin-specific reaginic antibodies. These observations suggest insulin, but not proinsulin, as the mediator of the immediate insulin allergic reaction.