Chain Of Human Insulin Research Articles

Insulin glargine: pharmacokinetic and pharmacodynamic basis of clinical effect

Glargine became the first long-acting insulin analogue. Glargine was designed to meet basal insulin requirements throughout the day with a single injection. Pharmacokinetics of insulin glargine is characterized by biotransformation into metabolites M1 and M2 that transforms the B chain of glargine so it is similar to the B chain of human insulin. Plasma concentrations of active M1 and M2 metabolites have no pronounced peaks during the day, resulting in lower glucose variability and hypoglycaemia risk when compared with NPH insulin. The metabolic activities of M1 and M2 metabolites are similar to the effect of glargine, whereas the mitogenic effects of these metabolites do not exceed the effect of human insulin. Insulin glargine shows a higher affinity for the insulin-like growth factor-1 (IGF-1) receptor when compared with human insulin. Glargine has no proliferative effect in vivo owing to its rapid conversion into metabolites. Pharmacokinetic and pharmacodynamic variability of glargine is comparable to other insulins. These characteristics are important for the clinical efficacy and safety of glargine.

Construction of a recombinant human insulin expression vector for mammary gland-specific expression in buffalo (Bubalus bubalis) mammary epithelial cell line.

The aim of the present study was construction of mammary gland specific expression vector for high level of human insulin (hINS) expression in transgenic buffalo for therapeutic use. We have constructed mammary gland specific vector containing human insulin gene and there expression efficiency was checked into in vitro cultured buffalo mammary epithelial cells (BuMECs). Human pro-insulin coding region was isolated from human genomic DNA by intron skipping PCR primer and furin cleavage site was inserted between B-C and C-A chain of human insulin by overlap extension PCR. A mammary gland-specific buffalo beta-lactoglobulin promoter was isolated from buffalo DNA and used for human insulin expression in BuMEC cells. The construct was transfected into BuMECs by lipofection method and positive transgene cell clones were obtained by G418 selection after 3 weeks. Expression of hINS in transfected cells were confirmed by RT-PCR, Immunocytochemistry, Western Blotting and ELISA. The pAcISUBC insulin-expressing clones secreted insulin at varying levels between 0.18 - 1.43 ng/ml/24 h/2.0 × 10(6) cells.

Diagnosis of Hypoglycemia due to Over-Dosage of Insulin Analog, Insulin Lispro

Serum insulin assay is used for the diagnosis of diabetes and hypoglycemia and is also used to quantify insulin for insulin pharmacokinetic evaluations. Serum C-peptide is usually measured in subjects who use insulin for evaluation of intrinsic insulin secretion. We experienced a hypoglycemic diabetic patient treated by premixed insulin lispro 75/25 (75% insulin lispro protamine suspension and 25% insulin lispro) who showed undetected serum levels of both insulin and Cpeptide. A 74-year-old type 2 diabetic man has been treated by injection of 26 units of premixed insulin lispro 75/25 before breakfast. In December 2010, his food intake decreased due to appetite loss, however, he injected 26 units of premixed insulin lispro 75/25, and subsequently developed disturbance of consciousness. He was admitted to our department, and his blood glucose level was 37 mg/dL. His consciousness promptly recovered after intravenous injection of glucose, and then he has been diagnosed as having hypoglycemia. We expected that his serum insulin level is high. However, serum insulin and also C-peptide were not detected. HbA1c is 5.5%, and anti-glutamic acid decarboxylase antibody was negative, and 125I-insulin binding rate was not too high (2.6%). His urinary C-peptide level was very low (4.0 μg/ day; normal range, 29.2 167.0 μg/day), suggesting the presence of severely decreased insulin secretion capacity. What is the cause of his hypoglycemia? Insulin analogs that are prepared with recombinant DNA technology are available for clinical use. One obvious question with insulin analogs is whether they are detectable by immunoassay. Serum insulin could not be detected by the chemiluminescent enzyme immunoassay (CLEIA) using Lumipulse Presto Insulin (Fuji Rebio, Tokyo, Japan) in our patient (Table 1). However, serum insulin could be measured by radioimmunoassay (RIA) using Insulin Eiken (Eiken Chemical Company, Tokyo, Japan). Lumipulse Presto Insulin could detect NPH human insulin, but, could not detect insulin lispro, and showed very low cross-reactivity with premixed insulin lispro 75/25 (Table 1). Insulin Eiken showed a high cross-reactivity with NPH human insulin, insulin lispro and premixed insulin lispro 75/25 (Table 1). Insulin lispro is a human insulin analog created by reversing the amino acids at positions 28 (Pro to Lys) and 29 (Lys to Pro) of the B chain of human insulin [1]. A sensitive RIA that is specific for insulin lispro has been developed [2]. Further, the combination of insulin assays that detect human insulin only or both human insulin and insulin analogs provides a new tool for studying pharmacokinetics of insulin lispro [3]. However, all of these assays are used mainly for research and are not usually used in clinical laboratories. Owen WE, et al studied cross-reactivity of insulin analogs with commercial insulin immunoassays [4]. Insulin lispro had a high cross-reactivity of 80% and 90% with the Access analyzer (Beckman Coulter) and the Advia Centaur analyzer (Bayer Diagnostics), respectively, whereas insulin lispro had a low cross-reactivity of 43% and 28% with the Coat-ACount (Diagnostic Products Corporation) and the IMMULITE 2000 analyzer (Diagnostics Products Corporation), respectively. The E170 method did not detect insulin lispro, indicating that the antibody used in this assay could not recognize an epitope that includes B28 and/or B29 of the B chain. The antibody in the assay of Lumipulse Presto InsuManuscript accepted for publication April 6, 2012

Expression of cholera toxin B subunit and the B chain of human insulin as a fusion protein in transgenic tobacco plants

A DNA construct containing the cholera toxin B subunit (CTB) gene genetically fused to a nucleotide sequence encoding three copies of tandemly repeated diabetes-associated autoantigen, the B chain of human insulin, was produced and transferred into low-nicotine tobaccos by Agrobacterium. Integration of the fusion gene into the plant genome was confirmed by polymerase chain reaction (PCR). The results of immunoblot analysis verified the synthesis and assembly of the fusion protein into pentamers in transgenic tobacco. GM1-ELISA showed that the plant-derived fusion protein retained GM1-ganglioside receptor binding specificity. The fusion protein accounted for 0.11% of the total leaf protein. The production of transgenic plants expressing CTB-InsB3 offers a new opportunity to test plant-based oral antigen therapy against autoimmune diabetes by inducing oral tolerance.

Anomalous mobility of sulfitolysed proteins in SDS-PAGE. Analysis and applications.

Some cysteine-containing proteins upon sulfitolysis have been found to show anomalously retarded SDS-PAGE mobilities in non-reducing gels. These proteins include bovine serum albumin, ovalbumin, aldolase, ribonuclease and a recombinant fusion protein (XA) consisting of a portion of gamma-interferon linked to the A chain of human insulin. This mobility shift has been employed to determine the stability of the sulfonated products and to study the kinetics of the sulfitolysis reaction. Partially sulfonated products of intermediate shifts were observed at 0.01% beta-ME, while 0.05% beta-ME gave a shift characteristic of the completely reduced protein. The undiluted sulfitolysis reagent reacted with XA to give within 1 min a gel shift characteristic of the fully sulfitolysed protein. Its transition stages could be visualized at 15, 30 and 60 min when the reagent was diluted four-fold. In the presence of 8 M urea, the sulfitolysis of BSA was nearly complete at 30 min when the sulfitolysis reagent was used at a dilution of 1:5. However, under the same conditions BSA was predominantly unsulfitolysed in the absence of urea. In order to elucidate the mechanism of sulfonation shift, several derivatives of XA, e.g. performic acid oxidized, alkylated with (a) iodoacetamide and (b) iodoacetate, have been prepared. While the mobility of XASSO3- was sensitive to the presence of beta-ME, all other derivatives moved in a beta-ME-insensitive fashion. Furthermore, while the nonreducing mobilities of the acidic derivatives (-SSO3-, -SO3- and -SCH2CO2-) were anomalously retarded and identical, the mobility of the iodoacetamide derivative was intermediate between the retarded acidic derivatives above and XA below. These studies have suggested a role of the extended conformation of the A chain of insulin in causing a mobility shift of the acidic derivatives in this series. Similar results were observed in an analogous series of derivatives prepared from BSA. Non-denaturing gel filtration analyses of native vs. sulfitolysed samples of serum albumin, ovalbumin and ribonuclease have indicated that the sulfitolysed proteins elute earlier than their native counterparts and appear to be significantly larger than their true molecular weights. Circular dichroism analysis has indicated significant loss in helicity of sulfitolysed BSA. This suggests that the retarded mobility of sulfitolysed proteins seen on SDS-PAGE is likely to be due to an expansion in the hydrodynamic volumes of these proteins, a phenomenon triggered by cleavage of disulfide bonds and further accentuated by the introduction of strongly negatively charged sulfonates.

Synthesis of a tetrapeptide using trimethylsilyl amino components

1. We have synthesized tetrapeptide (5–8) of the B chain of human insulin by using trimethylsilyl derivatives of the amino acids and peptides together with their alkyl esters. 2. Peptides can be prepared without racemization and in high yields by the use of the trimethylsilyl derivatives of the amino components.

Synthesis of human [9-leucine-B] Insulin.

The synthesis and isolation in purified form of [Leu9-B]insulin, a biologically active analogue of human insulin, is described. This analogue differs from the parent molecule in that the polar residue, serine, occupying position 9 in the B chain and located on the outside of the insulin molecule, has been replaced with the hydrophobic leucine residue. For the synthesis of this analogue the [Leu9]B chain of human insulin was chemically synthesized by the fragment condensation approach and isolated in the S-sulfonated form. Combination of this compound with the sulfhydryl form of human A chain afforded [Leu9-B]insulin. Separation of this analogue from the combination mixture and isolation as the hydrochloride in purified form were accomplished by chromatography on a carboxymethylcellulose column with an acetate buffer (pH 3.3) and an exponential sodium chloride gradient. [Leu9-B]Insulin possesses a potency of 13-14 IU/mg when assayed by the mouse convulsion method and 11-12 IU/mg by the radioimmunoassay method as compared to 23-25 IU/mg possessed by the natural hormone.

Tactics for simplification of purification process in the solid phase peptide synthesis.

These tactics depend primarily on coupling of a highly polar compound, as a highly polar handle in the purification process, with the terminal amino group of the desired sequence assembled on a solid support. Lysine was used as the highly polar compound in this study. All protecting groups labile towards hydrogen fluoride were removed as usual and the resulting crude peptide was purified through a carboxymethyl cellulose column. The amino terminal lysine residue was removed by Edman degradation to obtain the desired peptide. The technique thus developed has permitted the syntheses of peptide fragments of the B chain of human insulin, H-Gly-Phe-Phe-Tyr-Thr-Pro-Lys (Tfa)-Thr-OH and H-Gly-Ser-His-Leu-Val-OH, with a simple purification process in good yield and in high purity.

Insulin peptides. Part XXIII. The synthesis of a hexadecapeptide derivative related to the B chain of human insulin

A protected hexadecapeptide with the amino-acid sequence found at the C-terminal region of the human insulin B chain has been prepared by condensation of N-terminal hexapeptide and C-terminal decapeptide fragments, which were prepared stepwise. All the protecting groups employed are labile to hydrogen fluoride.

Insulin peptides. XVI. The synthesis of a nonapeptide and a dodecapeptide derivative related to the a chain of human insulin (positions 1-9 and 10-21).

Syntheses are described of protected peptides related to the N-terminus and C-terminus of the human insulin A chain. Thus the preparation is given of N-carbobenzox yglycyl-L-isoleucyl-L-valyl-7-f-butyl-L-glutamyl- L-glutaminyl-S-benzyl-L-cysteinyl-S-benzyl-L-cysteinyl-L-threonyl-L-serine hydrazide and N-carbobenzoxy-L-iso- leucyl-S-benzyl-L-cysteinyl-L-seryl-L-leucyl-L-tyrosyl-L-glutaminyl-L-leucyl-L-glutamyl- L- asparaginyl- L- tyrosyl - S - benzyl-L-cysteinyl-L-asparagine p-nitrobenzyl ester. The former derivative corresponds to positions 1-9 and the latter to positions 10—21 in the human insulin A chain sequence. R ecent communications3'4 from this laboratory described the synthesis of peptide derivatives related to the N-terminus of the A chain of sheep insulin and the synthesis of a dodecapeptide derivative con­ taining the C-terminal sequence of that chain. An in­ termediate in the construction of the latter peptide fragment, namely, the C-terminal nonapeptide portion, embodies the amino acid sequence found at the carboxyl terminus of the A chain of human insulin. The present report describes the synthesis of a protected nonapep­ tide and a protected dodecapeptide containing the N- terminal and C-terminal amino acid sequence, respec­ tively, of the human insulin A chain. The amino acid sequence of the reduced A chain of human insulin as determined by Nicol and Smith6 is glycylisoleucylvalylglutamylglutaminylcysteinylcyste- inylthreonylserylisoleucylcysteinylserylleucyltyrosylgul- taminylleucylglutamylasparaginyltyrosylcysteinylaspar- agine. An identical sequence was proposed for the reduced A chain of porcine insulin.6 The total synthesis of the human insulin A chain in the S-sul- fonated form is presented in the following paper7 and the preparation of two key intermediates used for that synthesis is described in the present report. One of these intermediates is the hydrazide of N-carboben- zoxyglycyl-L-isoleucyl-L-valyl - 7 - / - butyl -L - glutamyl - L - glutaminyl-S-benzyl-L-cysteinyl-S-benzyl-L-cysteinyl-L- threonyl-L-serine (I), the nonapeptide fragment which contains the amino acid sequence found at the amino terminus of the A chain, and the other intermediate is N - carbobenzoxy - L - isoleucyl - S - benzyl - L - cysteinyl - L - seryl-L - leucyl - L - tyrosyl - L - glutaminyl - L - leucyl - L - glu - tamyl-L-asparaginyl-L - tyrosy 1- S - benzyl - L - cysteinyl -L- asparagine ^-nitrobenzyl ester (XVI), the dodecapeptide

Insulin peptides. XI. The synthesis of the B chain of human insulin and its combination with the natural a chain of bovine insulin to generate insulin activity.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTInsulin Peptides. XI. The Synthesis of the B Chain of Human Insulin and Its Combination with the Natural A Chain of Bovine Insulin to Generate Insulin Activity1Panayotis G. Katsoyannis, Andrew M. Tometsko, James Z. Ginos, and Manohar A. TilakCite this: J. Am. Chem. Soc. 1966, 88, 1, 164–166Publication Date (Print):January 1, 1966Publication History Published online1 May 2002Published inissue 1 January 1966https://doi.org/10.1021/ja00953a032RIGHTS & PERMISSIONSArticle Views72Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (433 KB) Get e-Alerts Get e-Alerts