Syntheses of oligopeptides related to the insulin sequence B 22-25 (Arg-Gly-Phe-Phe) (author's transl)]

Syntheses of peptides with the sequences Gly-Phe, Gly-Phe-Phe, Arg-Gly-Phe and Arg-Gly-Phe-Phe are described. They were performed with the free acids, methyl esters and caramides. The peptides correspond partially or directly to the insulin sequence B 22 - 25 (Arg-Gly-Phe-Phe), the tetrapeptide amide or tetrapeptide methyl ester of which shows insulin-like activity (l.c.[1,2]). For testing the structural specificity of the arginyl residue, the following peptides were also synthesised: NG-NO2-Arg-Gly-Phe-Phe-NH2 and -OMe, Orn-Gly-Phe-Phe-NH2 and Cit-Gly-Phe-Phe--NH2. In connection with the above, the syntheses of the new derivatives Nalpha,Ndelta-Z2-L-ornithine p-nitrophenyl ester and N-Boc-L-citrulline p-nitrophenyl ester are described. All peptides were synthesised conventionally.     

Glucocorticoid-induced changes in insulin secretion related to the metabolism and ultrastructure of pancreatic islets

The effect of adrenalectomy and dexamethasone-treatment on insulin secretion was studied and related to the changes observed in the glucose oxidation, calcium uptake, cAMP and insulin content, as well as the ultrastructure of pancreatic rat islets. It was found that adrenalectomy was followed by a decreased glucose-induced insulin secretion, glucose oxidation, calcium uptake, cAMP and insulin content without any remarkable change observed at the ultrastructural level. Conversely, adrenalectomized-rats supplemented with dexamethasone showed an increased glucose-induced insulin secretion, glucose oxidation, calcium uptake and cAMP content but a diminished islet insulin content. At the ultrastructural level, a clear picture of increased secretory activity was found, with diminished number of mature B granules and greater number of pale granules, while rough endoplasmic reticulum and Golgi complex frequently appeared hypertrophic. These changes were only observed in the B cells. On account of our results, we might suggest that insulin secretion is partially controlled by glucocorticoid circulating levels throughout their effect on pancreatic islet metabolism.     

Structure and activity of insulin, XV[1-5]. Further evidence for the importance of arginine residue B22 in the activity of insulin. Semisyntheses of despentapeptide-(B23 - 30)-insulins varied in B22 using desnonapeptide-(B22 - 30)-insulin and tetrapeptides

Insulin hexamethyl ester was digested by trypsin. The resulting desoctapeptide-(B23 - 30)-insulin pentamethyl ester was purified. This compound was digested by carboxypeptidase B to remove the arginine residue B22 at the end of the B chain. Then the N-terminal amino groups of the remaining desnonapeptide-(B22 - 30)-insulin pentamethyl ester were protected with the Boc residue. The free carboxyl group of the glutamic acid residue B21 of this product was coupled to the following synthetic tetrapeptide esters: Arg-Gly-Phe-Phe-OMe, Lys(Boc)-Gly-Phe-Phe-OMe, Orn(Boc)-Gly-Phe-Phe-OMe, Cit-Gly-Phe-Phe-OMe, Ala-Gly-Phe-Phe-OMe and Gly-Gly-Phe-Phe-OMe. The syntheses of these peptide esters are described. After removal of all protecting groups, despentapeptide-insulin (B22-Arg) and analogues of this product with variation in position B22 could be obtained. They were purified by column chromatography. The biological activities of these components were determined by the mouse fall test. In the case of despentapeptide insulin (C-terminus Arg-Gly-Phe-Phe), the activity rose to the expected value of 34%. The insulin variants with amino acid residues other than arginine in position B22 had much lower activities: with lysine 13%, with ornithine 12%, with citrulline 9%, with alanine 8% and with glycine 6%. Desnonapeptide-insulin by itself posses an activity of 3%. These results demonstrate once more the essential nature of arginine residue B22 for insulin activity.     

B22 Glu Des-B30 Insulin： A Novel Monomeric Insulin

Studies on monomeric insulin with reduced self-association are important in the development of insulin pharmaceutical preparations with rapid hypoglycemic action on patients with diabetes. Here we report a novel monomeric insulin, B22 Glu des-B30 insulin, prepared from a single chain insulin precursor with B22 Arg mutated to Glu, which was expressed in Pichia pastoris and converted to B22 Glu des-B30 insulin by tryptic digestion. It still retains 50% of the in vivo biological activity of porcine insulin and does not form a dimer even at a concentration of 10 mg/ml, showing that B22 Glu plays a key role in reducing the self- association of the insulin molecule without greatly reducing its biological activity. This novel monomeric insulin might have potential applications in the clinic.     

Antagonism of insulin action on muscle by the albumin-bound B chain of insulin

1. The presence of a substance associated with human albumin that exerts anti-insulin activity on the isolated rat diaphragm has been confirmed. This factor has been removed from albumin, thereby providing a source of non-antagonistic carrier protein. 2. Derivatives of the polypeptide B chain of insulin obtained by chemical scission of the interchain disulphide bonds have been separated by conventional techniques. In the presence of non-antagonistic albumin, the reduced and sulpho-B chain preparations inhibited insulin action on muscle. 3. The B chain resulting from reductive cleavage of insulin by bovine-liver extracts, in association with human albumin, exhibited a comparable anti-insulin effect. 4. It is postulated that the B chain interacts with albumin to enable solubilization of the chain and that inhibition of insulin action on muscle may occur as a result of competition for cellular receptor sites by the B chain. 5. The implication of these findings in relation to a circulating insulin antagonist is discussed.     

Dogfish insulin. Primary structure, conformation and biological properties of an elasmobranchial insulin

Insulin from an elasmobranch, the spiny dogfish (Squalus acanthias) has been purified to near homogeneity by means of acid-ethanol extraction and salt precipitation. The amino acid sequences of the performic-acid-oxidised A and B chains have been determined and exhibit some unusual features. The A chain contains a total of 22 amino acids; only the insulin from coypu (a member of the Rodentia suborder, Hystricomorpha), has previously been reported to contain an extension past the A21 asparagine. The B10 histidine, which is involved in the formation of the insulin hexamers in higher vertebrates through the co-ordination of zinc, is present in this elasmobranch insulin. Several substitutions relative to bovine insulin occur in the proposed receptor binding region (A5Gln leads to His, B21Glu leads to Pro, B22Arg leads to Lys, B25Phe leads to Tyr). In spite of these substitutions, the maximal response in the rat epididymal fat cell assay is the same for bovine and dogfish insulins; the concentration required to produce the half-maximal response is, however, approximately threefold greater for dogfish insulin than that of bovine insulin. The use of interactive computer graphics model-building predicts that the dogfish insulin can attain a three-dimensional structure very similar to that of bovine insulin; circular dichroic spectra are presented which support the model-building studies.     

Shortened insulin analogues: marked changes in biological activity resulting from replacement of TyrB26 and N-methylation of peptide bonds in the C-terminus of the B-chain

The role of three highly conserved insulin residues PheB24, PheB25, and TyrB26 was studied to better understand the subtleties of the structure-function relationship between insulin and its receptor. Ten shortened insulin analogues with modifications in the beta-strand of the B-chain were synthesized by trypsin-catalyzed coupling of des-octapeptide (B23-B30)-insulin with synthetic peptides. Insulin analogues with a single amino acid substitution in the position B26 and/or single N-methylation of the peptide bond at various positions were all shortened in the C-terminus of the B-chain by four amino acids. The effect of modifications was followed by two types of in vitro assays, i.e., by the binding to the receptor of rat adipose plasma membranes and by the stimulation of the glucose transport into the isolated rat adipocytes. From our results, we can deduce several conclusions: (i) the replacement of tyrosine in the position B26 by phenylalanine has no significant effect on the binding affinity and the stimulation of the glucose transport of shortened analogues, whereas the replacement of TyrB26 by histidine affects the potency highly positively; [HisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide and [NMeHisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide show binding affinity 529 and 5250%, respectively, of that of human insulin; (ii) N-methylation of the B24-B25 peptide bond exhibits a disruptive effect on the potency of analogues in both in vitro studies regardless the presence of amino acid in the position B26; (iii) N-methylation of the B23-B24 peptide bond markedly reduces the binding affinity and the glucose transport of respective analogue [NMePheB24]-des-tetrapeptide (B27-B30)-insulin-B26-amide.     

Potential mechanism of insulin action on glucose transport in the isolated rat diaphragm. Apparent translocation of intracellular transport units to the plasma membrane

[3H]Cytochalasin B binding and its competitive inhibition by D-glucose have been used to quantitate the number of functional glucose transport units in plasma and microsomal membranes prepared from intact rat diaphragm. In a series of three experiments, plasma membranes prepared from diaphragms which have not been incubated with insulin bind approximately 16 pmol of cytochalasin B/mg of membrane protein to the D-glucose-inhibitable binding site. If 280 nM (40,000 microunits/ml) insulin is present during the incubation, cytochalasin B binding to the plasma membranes is increased approximately 2-fold without alteration in the dissociation constant of this site. Membranes in the microsomal fraction prepared from diaphragms which have been incubated for 30 min in the absence of insulin contain 21 pmol of D-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. However, in the presence of insulin during the incubation period, the number of these sites in the microsomal fraction is decreased to 12 pmol/mg of membrane protein. These results suggest that insulin stimulates glucose transport in the isolated rat diaphragm primarily through a translocation of functional glucose transport units from an intracellular membrane pool to the plasma membrane. These results are similar to the results observed in rat adipose cells (Cushman, S. W., and Wardzala, L. J. (1980) J. Biol. Chem. 255, 4758-4762) and suggest that this mechanism of insulin-stimulated glucose transport activity may be general to other cell types.     

Effects of hexapeptide, a compound analogous to insulin B chain fragment B-21-26(DP-432) on the glucose uptake into the perfused hind limb of rats

DP-432 is a synthetic new peptide analogous to insulin B-chain fragment B21-26. It has been reported that this hexapeptide shows insulin potentiating action besides insulin-like activities in adipose tissue and diaphragm of rats. The perfusion of the hind limb of rats was performed according to the procedure of Ruderman with some modifications. The medium was Krebs-Ringer bicarbonate buffer containing 0.5% bovine albumin and 8.3 mM glucose. The erythrocytes were omitted from the medium. In this experimental procedure it was shown that the glucose uptake into the hind limb of rats increased by infusion of DP-432 (100 microgram/ml). The amount of glucose uptake into the hind limb responded to insulin doses (50, 100, 1000 muU/ml). DP-432 had insulin-like activity of 100 muU/ml. However, DP-432 itself did not have any potentiation of insulin activity on the muscle.     

Purification and characterization of a rat liver cytosol neutral thiol peptidase that degrades glucagon, insulin, and isolated insulin A and B chains

A thiol peptidase that catalyzes at near neutral pH the hydrolysis of insulin, the isolated A and B chains of insulin, and glucagon was purified from rat liver cytosol by fractionation on Sephadex G-200, Affi-Gel Blue, and Spherogel TSK-G 3000 SW. The purified enzyme showed a single component by chromatography on a Spherogel TSK column and by gel filtration on a Sephadex G-200 column. The native enzyme has a molecular weight of approximately 180,000 and consists of two subunits having pI's of 5.9 and 6.3. Studies on its substrate specificity showed that the purified enzyme degrades glucagon, insulin, insulin B chain, and insulin A chain, but it does not degrade proinsulin, ACTH, or denatured hemoglobin. Kinetic analyses were performed on three substrates. The Km values were: 34 nM for insulin, 276 nM for insulin B chain, and 3.5 microM for glucagon. The kcat and Vm/Km values were glucagon greater than B chain greater than insulin. Thus, the enzyme 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. The effect of several potential activators and inhibitors on the enzyme's activity was investigated. The enzyme activity was markedly inhibited by N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and Np-tosyl-L-phenylalanine chloromethyl ketone (TPCK), and was partially inhibited by dithiothreitol, by the chelating agents EDTA and EGTA, and by phenylmethylsulfonyl fluoride (PMSF). Bacitracin inhibited the activity of the enzyme, but the protease inhibitors aprotinin, leupeptin, pepstatin, and phosphoramidon had little or no effect. Reduced glutathione, iodoacetate, and N alpha,p-tosyl-L-lysine chloromethyl ketone (TLCK) also had little or no effect on the enzyme activity.     

(A1-D-alanine) insulin (author's transl)]

Starting from porcine insulin, A1-glycine was substituted by D-alanine and by L-alanine for comparison. Replacement of A1-glycine by L-alanine revealed the known decrease in the biological activity. [A1-D-Alanine]insulin, however, has the same blood sugar lowering activity as insulin and is slightly more active in its influence on the glucose uptake into the rat diaphragm. The specific binding to insulin receptors of rat liver is decreased as, compared to insulin, but increased as compared to the L-alanine analogue.     

Synthesis of an insulin-like compound consisting of the A chain of insulin and a B chain corresponding to the B domain of human insulin-like growth factor I

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.     

Comparative study of four arginine ester hydrolases, ME-1, 2, 3 and 4 from the venom of Trimeresurus mucrosquamatus (the Chinese habu snake)

Four arginine ester hydrolases, ME-1, 2, 3 and 4 from the venom of Trimeresurus mucrosquamatus had been isolated and characterized by Sugihara et al. (1980, 1981, 1982, 1983). Immunologically, ME-1, 2, 3 and 4 are identical. The four enzymes hydrolyzed Pro-Phe-Arg-MCA and z-Phe-Arg-MCA. Furthermore, ME-2 slightly hydrolyzed Boc-Val-Pro-Arg-MCA, Boc-Phe-Ser-Arg-MCA and Boc-Ile-Glu-Gly-Arg-MCA. ME-1 cleaved almost simultaneously the Arg(22)-Gly(23) and Phe(25)-Tyr(26) bond of oxidized insulin B chain. ME-2 and 3 also hydrolyzed the same bond of insulin B chain, but the activity was not as potent as ME-1. ME-4 did not cleave the substrate. The four enzymes hydrolyzed C-terminal of arginine in the biologically active peptides. Four arginine ester hydrolases showed fibrinogenolytic activity. ME-1 and 2 first cleaved B beta-chain and then A alpha-chain. On the contrary, ME-3 and 4 cleaved A alpha- and B beta-chain simultaneously. The four enzymes also hydrolyzed fibrinogen in plasma cleaving B beta- and gamma-chain and slightly digesting A alpha-chain. The various inhibitors affected TAME (tosyl-arginine-methylester) and the fibrinogen hydrolytic activity of the four enzymes. All four enzymes had fibrinolytic activity.     

Immunohistochemical studies of insulin receptors in the liver

The indirect peroxidase-labeled antibody method was used to localize Insulin Receptors (IR) in rat livers. For experiments the rabbit antisera against synthetic peptides correspond to the deduced amino acid sequence of alpha- and beta-subunits of human placenta insulin receptors were used as the first sera. Our results have shown that 30 min after intraperitoneal and intraventricular injections of I U insulin in controls and MSG-treated animals the staining of DAB reaction products in livers became stronger as compared to those injected with saline solution. After intraperitoneal and intraventricular injections of insulin the electron-microscopic investigations of rat livers have found the internalization of IR in hepatocytes by interaction of receptors with exo- and endogenous insulin.     

B22-D-arginine]insulin: synthesis and biological properties

An analogue of porcine insulin which differs from the native molecule in that the amino-acid residue B22-L-arginine is replaced by its D-enantiomer has been synthesized. The [D ArgB22]B-chain was synthesized by the segment condensation method and purified as the di-S-sulfonate by ion exchange chromatoggraphy on SP-Sephadex at pH 3.5. Combination with native porcine sulfhydryl A-chain gave [DArgB22]insulin which was purified by ion exchange chromatography on SP-Sephadex at pH 4.5 with a linear NaCl gradient. The biological activity of this analogue as measured by glucose oxidation in rat epididymal adipocytes was 2%. Thymidine incorporation into DNA of human fibroblast was 16%. The immunoreactivity using antipork insulin antibody in a double antibody immunoassay was 4%. The receptor-binding affinity as measured by radioreceptor assays was 2% with cultured human fibroblasts and 1% with rat adipocytes. These results suggest that the L-configuration at B22-arginine is essential for retaining the biological, immunological and receptor-binding properties of the hormone.     

Synthesis and biological activity of seventeen analogues of human insulin

We synthesized seventeen analogues of human insulin, applying the principle of stepwise, selective formation of the disulphide bonds. Most of these analogues only differ from human insulin in the replacement of a single amino acid in positions 2, 5, 6, 7, 8 and 11 of the A chain and 5, 7, 13 and 16 of the B-chain. The influence of these modifications on the physicochemical properties of the analogues is discussed. Eight analogues could be crystallized. All the analogues produce the same biological effects as insulin, but differ markedly in their potency. In isolated fat cells in vitro, [HisA8]insulin showed a relative potency of 2.46 in stimulating glucose oxidation (human insulin = 1), whereas [D-CysA6,A11]insulin had a potency of only 0.00027. Very low potency was observed when IleA2 or the half-cystines A6, A7, A11 or B7 were modified. Replacement of the invariant GlnA5 by alanine only reduced potency slightly. All the analogues are full agonists. The effects of the analogues on glucose oxidation and lipolysis are correlated, supporting the view that they are mediated by a common receptor on the fat-cell membrane. Hypoglycaemic potencies in the rat were similar to potencies in vitro. As expected, no correlation was demonstrable between antiserum binding--measured in the radioimmunoassay--and biological activity. Several results of this investigation are difficult to reconcile with the current view regarding the structure-activity relationship of insulin which appears to require further refinement.     

Purification and substrate specificity of two cysteine proteinases of Giardia lamblia.

The proteinase activity present in homogenates of trophozoites of Giardia lamblia, active on azocasein and urea-denaturated hemoglobin, was separated into two different enzymes by a series of purification procedures. These procedures included gel filtration on Fractogel TSK HW-55 (F), organomercurial agarose affinity chromatography, and ion exchange chromatography on DEAE-cellulose. By chromatography on Sephadex G-100, two purified enzymes exhibited relative molecular weights of Mr = 95,000 and 35,000 +/- 10%, respectively. On the basis of inhibition by thiol reagents and abrogation of this effect by dithiothreitol and cysteine, they were identified as cysteine proteinases. Proteinase I (Mr = 95,000) and proteinase II (Mr = 35,000) were active against the beta-chain of insulin releasing characteristic fragments. However, differences in substrate specificities of the two enzymes could be observed by using synthetic peptides that represent sequences 1-6, 8-18, and 20-30 of the insulin beta-chain. Furthermore, the synthetic tetrapeptides Arg-Gly-Phe-Phe, Arg-Gly-Leu-Hyp, and Arg-Arg-Phe-Phe were hydrolyzed by the two proteinases releasing Phe-Phe and Leu-Hyp, respectively. Compared with Arg-Gly-Phe-Phe, the rates of hydrolysis of Arg-Gly-Leu-Hyp and Arg-Arg-Phe-Phe at substrate concentrations of 1 mM were 91% and 63% (proteinase I) and 80% and 57% (proteinase II), respectively.     

21-arginine-A] insulin: a biologically active analog.

An analog of sheep insulin which differs from the parent molecule in that the C-terminal amino acid residue of the A chain, asparagine, is replaced by arginine, has been synthesized and isolated in highly purified form. The [Arg21] A chain of sheep insulin was synthesized by the fragment condensation approach and isolated as the S-sulfonated derivative. Conversion of the latter into the sulfhydryl form and interaction with the S-sulfonated B chain of bovine (sheep) insulin yielded [Arg21-A] sheep insulin, which was purified by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. The [Arg21-A] sheep insulin shows potencies of 10.5--12.5 IU/mg when assayed by the mouse convulsion method and 8.6 IU/mg by the radioimmunoassay method (cf. 23--25 IU/mg for the natural hormone). It has been suggested that in the insulin molecule the A21 asparagine participates in salt bridge- and hydrogen bond-forming interactions which are critical in the biological activity of the hormone. Although the [Arg21-A] analog still retains these interactions, it is only ca. 50% as active as the natural hormone. It is speculated that other factors than the above mentioned interactions come into play, which involve the side chain of the A21 amino acid residue and affect the biological activity of the hormone.     

Molecular recognition between insulin and dextran encapsulated gold nanoparticles

Insulin is a peptide hormone that can regulate the metabolism of carbohydrates and lipids. This hormone is closely related to glucose‐uptake in cells and can control blood glucose levels. Dextran is a polysaccharide composed of glucose units. In this study, we discovered that dextran‐encapsulated gold nanoparticles (AuNPs@Dextran) and nanoclusters (AuNCs@Dextran) can be used to recognize insulin. The dissociation constant of insulin toward AuNPs@Dextran was estimated to be ～5.3 × 10^?6 M. The binding site on insulin toward the dextran on the nanoprobes was explored as well. It was found that the sequence of numbers 1–22 on the insulin B chain can interact with the dextran encapsulated nanoprobes. Additionally, we also demonstrated that the dextran‐encapsulated nanoprobes could be used as concentration probes to selectively enrich trace amounts of insulin (～1 pM) from serum samples. Copyright © 2016 John Wiley & Sons, Ltd.