Vertebrate Insulin Research Articles

Insulin-induced effect on diapausing pupae of tropical tasar silkworm, Antheraea mylitta Drury (Lepidoptera: Saturniidae)

Diapausing pupae of tasar silkworm, Antheraea mylitta Drury were injected with ox pancreas insulin (40 I.U. per ml) on day 150 to observe the effect of this vertebrate hormone on diapause physiology. Insulin at the dose of 5 μl (0.2 I.U.) per pupa exhibited its positive impact by enhancing the incidence of male and female moth eclosion, female moth weight, egg production (laid + unlaid), and fecundity and shortening of time taken for emergence in both sexes. But, 10 μl (0.4 I.U.) dose of insulin increased only the female eclosion and reduced the time taken for emergence in each sex when compared to that of control. Administration of higher dose of insulin (10 μl per pupa) decreased the egg production and fecundity. No significant changes were observed in hatchability and incubation phenomena of eggs laid by the treated females. Further, both the doses of insulin were able to bring about the morphometric changes of testis and ovary by altering their shape and size during pupal diapause. Thus, it reveals from the preliminary findings that vertebrate insulin induces diapause physiology of A. mylitta.

The closely related neuropeptide genes encoding adipokinetic hormones I and II have very different 5'-flanking regions.

Adipokinetic hormones I and II are 10- and 8-amino-acid grasshopper neuropeptides that are derived from 63- and 61-amino-acid peptide precursors, respectively. Each precursor is encoded by a separate gene consisting of three very small exons separated by two large introns. The identical exon structure of the two genes suggests that they evolved through duplication of a common ancestral gene. Despite the precise conservation of exon structure and the similarity of the coding sequences, the two genes have very different 5'-flanking regions, suggesting that they are differentially regulated. For example, sequences similar to the vertebrate insulin enhancer elements NIR and FAR are present upstream of the promoter region of the adipokinetic hormone II gene, but not in the adipokinetic hormone I gene. Both of these insect genes contain short interspersed repetitive DNA sequences in their introns that may have facilitated a gene duplication event.

Paradoxical structure and function in a mutant human insulin associated with diabetes mellitus.

The solution structure of a diabetes-associated mutant human insulin (insulin Los Angeles; PheB24-->Ser) was determined by 13C-edited NMR spectroscopy and distance-geometry/simulated annealing calculations. Among vertebrate insulins PheB24 is invariant, and in crystal structures the aromatic ring appears to anchor the putative receptor-binding surface through long-range packing interactions in the hydrophobic core. B24 substitutions are of particular interest in relation to the mechanism of receptor binding. In one analogue ([GlyB24]insulin), partial unfolding of the B chain has been observed with paradoxical retention of near-native bioactivity. The present study of [SerB24]insulin extends this observation: relative to [GlyB24]insulin, near-native structure is restored despite significant loss of function. To our knowledge, our results provide the first structural study of a diabetes-associated mutant insulin and support the hypothesis that insulin undergoes a change in conformation on receptor binding.

Apterous, a gene required for imaginal disc development in Drosophila encodes a member of the LIM family of developmental regulatory proteins.

The apterous (ap) gene is required for the normal development of the wing and haltere imaginal discs in Drosophila melanogaster. ap encodes a new member of the LIM family of developmental regulatory genes. The deduced amino acid sequence of ap predicts a homeo domain and a cysteine/histidine-rich domain known as the LIM domain. In these domains ap is highly similar to the mec-3 and lin-11 proteins of Caenorhabditis elegans and to the vertebrate insulin enhancer-binding protein isl-1. ap is presumably required for transcriptional regulation of genes involved in wing and haltere development. The nature of the defects in homozygous null mutant flies is consistent with the pattern of ap expression in the larval imaginal discs. ap is also expressed in a complex pattern in the embryo, including portions of the peripheral nervous system (PNS) and central nervous system (CNS). A requirement for ap expression in the larval and adult CNS may be the underlying cause of the defects in hormone production and vitellogenesis described for ap mutations.

Demonstration of insulin-related substances in the central nervous systems of pulmonates and Aplysia californica

the occurrence of insulin-related substances in the central nervous system of pulmonates and Aplysia californica was investigated by means of immunocytochemistry and in situ hybridization. Previous experiments have shown that, in Lymnaea stagnalis, the growth hormone-producing neurons in the cerebral ganglia (the so-called light green cells) express at least 5 genes that are related to the vertebrate insulin genes, i.e., they encode prohormones that are composed of a B- and A-chain and a connecting C peptide. These insulin related molecules also have the amino acids essential for their tertiary structure (viz. cysteines) at identical positions to those of the vertebrate insulins. In the investigated basommatophoran and stylommatophoran snails and slugs, neurons reacted with an antiserum raised against the C peptide of one of the molluscan insulin-related peptides. These neurons can be considered to be, based on morphological and endocrinological criteria, homologous to the light green cells of L. stagnalis. In A. californica, all central ganglia contain immunoreactive neurons. The highest number (about 50) was observed in the abdominal ganglion. The present results indicate that insulin-related substances are generally occurring neuropeptides in the central nervous system of molluscs.

Physiology of fish endocrine pancreas

From the very beginning of physiological studies on the endocine pancreas, fish have been used as experimental subjects. Fish insulin was one of the first vertebrate insulins isolated and one of the first insulins whose primary and then tertiary structures were reported. Before a second pancreatic hormone, glucagon, was characterized, a physiologically active 'impurity', similar to that in mammalian insulin preparations, was found in fish insulins.Fish have become the most widely used model for studies of biosynthesis and processing of the pancreatic hormones. It seems inconceivable, therefore, that until the recent past cod and tuna insulins have been the only purified piscine islet hormones available for physiological experiments. The situation has changed remarkably during the last decade.In this review the contemporary status of physiological studies on the fish pancreas is outlined with an emphasis on the following topics: 1) contents of pancreatic peptides in plasma and in islet tissue; 2) actions of piscine pancreatic hormones in fish; 3) specific metabolic consequences of an acute insufficiency of pancreatic peptides; 4) functional interrelations among pancreatic peptides which differ from those of mammals. The pitfalls, lacunae and the perspectives of contemporary physiological studies on fish endocrine pancreas are outlined.

CDNA structure and expression of bombyxin, an insulin-like brain secretory peptide of the silkmoth Bombyx mori

Bombyxin, previously referred to as 4K-prothoracicotropic hormone, is a brain peptide of the silkmoth Bombyx mori, the amino acid sequence of which shows considerable homology with vertebrate insulin family peptides. Two independent clones have been isolated from a Bombyx larval brain cDNA library by using a synthetic oligonucleotide probe, one with the complete coding region for preprobombyxin (lambda Bb360) and the other covering the coding region, possibly for bombyxin, only partially (lambda Bb204). lambda Bb360 encodes preprobombyxin in the order of prepeptide/B-chain/proteolytic cleavage signal/C-peptide/proteolytic cleavage signal/A-chain. This domain organization of preprobombyxin is the same as that of preproinsulins, suggesting that the tertiary structure and posttranslational modification mechanism are conserved through the evolution of bombyxin and insulin. Genomic Southern hybridization analyses using this cDNA as probe suggest that the Bombyx genome contains multiple copies of bombyxin gene. Northern hybridization analyses indicate that the concentration of lambda Bb360-type bombyxin mRNA in the bombyxin-producing cells is remarkably high (2.8 x 10(9) molecules/micrograms of total RNA), without undergoing appreciable change during larval-pupal development.

A substance resembling somatomedin C in the American cockroach

Material antigenically resembling somatomedin C (type I insulin-like growth factor, IGF-I) is demonstrated in the American cockroach Periplaneta americana by means of a monoclonal antibody immunoperoxidase technique. It was localized histochemically in neuronal cell somata and axonal fibers (probably interneurons) of the central nervous/neuroendocrine system and in ‘endocrine-type’ cells lining the midgut epithelium. The IGF-I-like substance is different from vertebrate insulin and also distinct from materials immunostained by different insulin antibodies in the brain and neurohaemal complex of this insect species. These findings are viewed in the light of recent reports on the presence and action of insulin-like chemicals in insects, and with respect to the existence of an insect brain-midgut system similar to the mammalian brain-gastroenteropancreatic system.

Isolation and structure of lamprey ( Petromyzon marinus) insulin

Insulin has been purified to homogeneity from the caudal and cranial pancreas of the adult sea lamprey ( Petromyzon marinus). The final yield was 18.6 nmol/g (127.8 μg/g). The structures of both A- and B-chains have been determined using amino acid analyses, gasphase sequence analyses, and proteolytic mapping by fast atom bombardment-mass spectrometry. The sequence of the A-chain was found to be GIVEQCCHRKCSIYDMENYCN. The sequence of the B-chain, extended at the amino terminus, was determined to be SALTGAGGTHLCGSHLVEALYVVCGDRGFFYTPSKT. Lamprey insulin retains the common features of vertebrate insulins. Sea lamprey insulin has no more homology to hagfish ( Myxine glutinosa) insulin than it has to the teleost fish or to mammalian insulins.

Vertebrate insulin induces diapause termination in Pieris brassicae pupae

Due to its close structural homology with the 4K prothoracicotropic hormone isolated from Bombyx mori, we tested the ability of vertebrate insulin to break pupal diapause in a Lepidopteran, Pieris brassicae. Injection of 5μg of bovine insulin in diapausing pupae led to diapause termination and synchronous adult eclosion; the effect of insulin was dose-dependent. Bovine insulin-A chain and B chain injected separately failed to show any biological activity suggesting that the intact structure of the molecule is required. Bovine insulin also promoted adult development of decapitated diapausing animals. We show that insulin triggers a reactivation of the neuroendocrine system leading to a neosynthesis of ecdysone beginning 6 days after treatment. This neosynthesis also occurred in beheaded animals suggesting that insulin stimulates the prothoracic glands without acting via the brain.

Partial purification and characterization of an insulin-like material from spinach and Lemna gibba G3.

The existence in invertebrates, unicellular eukaryotes, and prokaryotes of materials that resemble several vertebrate peptide hormones led to the suggestion that these peptide messengers may have arisen earlier in evolution than had previously been thought. Consistent with this hypothesis, we describe here material in two plants, spinach and Lemma gibba G3, that is very similar to mammalian insulin, yet distinctive. In each of the early purification steps, which consisted of acidic methanol chloroform extraction and sequential chromatography on C-18 hydrophobic resin, Sephadex G-50, CM-Sepharose, and a short C-3 high performance liquid chromatography column, the immunoactive material from plants resembled the common vertebrate insulins. The protein nature of the material was suggested by its destruction by Pronase but not by the inactivated enzyme. In addition, on TSK chromatography it eluted in a position similar to that of insulin, i.e. equivalent to a protein of 6000 daltons. Using an isocratic high performance liquid chromatography system, the plant immunoactivity eluted earlier, and thus was more hydrophilic, than most of the common mammalian insulins, including pork insulin. The interaction of the plant material with anti-insulin antibodies in a radioimmunoassay was confirmed by using an affinity column of anti-insulin antibodies which adsorbed the plant immunoactivity at neutral pH, and released the material with acid elution. Using a quantitative double radioimmunoassay, the plant insulin-like material was distinguished immunologically from chicken insulin. Although the plant insulin-like material is clearly distinct from pork insulin chromatographically, and from chicken insulin immunologically, it resembles vertebrate insulins in its overall configuration. The plant insulin-like material bound to insulin receptors on IM-9 lymphocytes and stimulated glucose oxidation and lipogenesis in isolated adipocytes from young rats. The bioactivity was neutralized in the presence of anti-insulin antibodies, but not in the presence of normal guinea pig IgG. The role of this insulin-like material in plants is unknown but its existence is consistent with an early evolutionary origin of the insulin messenger peptide family. Alternatively we cannot exclude a later convergent development of this family or introduction of vertebrate DNA into plants.

Amino acid sequence of a prothoracicotropic hormone of the silkworm Bombyx mori

We have determined the complete amino acid sequence of 4K-PTTH-II, one of three forms of the M(r) 4400 prothoracicotropic hormone of the silkworm Bombyx mori, active to brainless pupae of Samia cynthia ricini. Like vertebrate insulin, it consists of two nonidentical peptide chains (A and B chains). The A chain consists of 20 amino acid residues. The B chain is a mixture of four microheterogeneous peptides, two of which consist of 28 residues, and the other two, of 26 residues. 4K-PTTH-II has considerable sequence homology (40%) with human insulin, and it resembles porcine relaxin both in the carboxyl-terminal cysteine residue of the A chain and in the amino-terminal pyroglutamic acid residue of the B chain. The identical distribution of the six cysteine residues also indicates that 4K-PTTH-II belongs to the insulin family.

Insulin-related material in microbes: similarities and differences from mammalian insulins.

We have reported that extracts of Escherichia coli and Tetrahymena grown in synthetic media contained material that reacted specifically in the immunoassay and bioassay for insulin. One additional strain of Tetrahymena and four of E. coli yielded amounts of material similar to those reported previously. In addition to their behavior on Sephadex G-50, the immunoactive insulin-related materials from the microbial sources behaved like authentic vertebrate insulins in their ability to be adsorbed to and eluted from disposable octadecasilylsilica cartridges, DEAE-Sephadex, DEAE-cellulose, and one system of high-pressure liquid chromatography (HPLC). As with less purified microbial material, the "insulin" that had been purified on DEAE and HPLC, when tested for its bioactivity, had an immunoactivity:bioactivity ratio of approximately unity and the bioactivity was largely neutralized by anti-insulin antibody. Because the material from the microbes was so similar to authentic insulins, studies were undertaken to demonstrate that inadvertent contamination with vertebrate insulins was highly unlikely. Blanks carried through the entire procedure were always negative. Tetrahymena grown and extracted in another laboratory gave the same results. Tetrahymena that had been grown but then allowed to stand in the fermenter under adverse conditions and then carried through the entire procedure were devoid of insulin. Tetrahymena that were homogenized and subjected to acid hydrolysis were devoid of insulin. Further substantiation that exogenous contamination was highly unlikely was provided by two other types of experiments. In one of these, it was shown that the subcellular distribution of exogenously added porcine insulin or porcine 125I-labeled insulin was different from the distribution of endogenous insulin. In the second type of experiment, it was shown that during the log phase of growth of Tetrahymena or of E. coli the insulin content of the system increased multifold in a fashion that might be expected for living organisms but quite unexpected for exogenous contamination. (Interestingly, the insulin content of the E. coli medium far exceeded that which might be contributed by death of cells, estimated by the content in the medium of an intracellular enzyme.) When E. coli was grown and processed in four other laboratories having no contact with our own, similar levels of insulin-related material were recovered.

Purification of insulin-like peptides from insect haemolymph and royal jelly

Insulin-like peptides from the haemolymph of the tobacco hornworm, Manduca sexta L. and from royal jelly of the honey bee, Apis mellifera L., were purified by acid extraction, ion-exchange chromatography, gel filtration and affinity chromatography. Quantities detected were 500 and 25 pg porcine insulin equivalents per gram of lyophilized material for haemolymph and royal jelly, respectively. M. sexta insulin was similar to vertebrate insulin in solubility, chromatographic, immunological and biological properties. Amino acid compositions were comparable except for serine, leucine and phenylalanine. The A. mellifera peptide had similar properties, but was not isolated in large enough quantity to determine its amino acid composition. These results demonstrated that insect insulin and vertebrate insulin are structurally similar.

Initial sites of insulin cleavage and stereospecificity of carboxyl proteinases from Aspergillus sojae and Pycnoporus coccineus

Initial cleavage sites of native insulin at a pH of about 3 and stereospecificity were investigated by fungal carboxyl proteinases (EC 3.4.23.6) from Aspergillus sojae, a species of fungi imperfecti, and Pycnoporus coccineus (formerly designated Trametes sanguinea), a wood deteriorating Basidiomycete, respectively. Fungal carboxyl proteinases were used as a model of vertebrate insulin degradation. A. sojae carboxyl proteinase I primarily hydrolyzed two peptide bonds located on the surface of native insulin monomer, the B16-B17 (Tyr-Leu) and B24-B25 (Phe-Phe) bonds, and secondarily the buried bonds, A15-A16 (Gln-Leu), B15-B16 (Leu-Tyr) and B14–B15 (Ala-Leu), at pH 3.2 and 30°C. The initial cleavage sites of A. sojae carboxyl proteinases I towards native insulin were not identical with the initial cleavage sites towards the oxidized B chain of insulin. P. coccineus carboxyl proteinase I a selectively hydrolyzed B14-B15 (Ala-Leu), B16-B17 (Tyr-Leu) and B24-B25 (Phe-Phe) bonds in the native insulin at pH 2.7. Based on these findings we suggest that the stereospecificity of the fungal carboxyl proteinases is similar to that of cathepsin D (EC 3.4.23.5), and that the synthesis and degradation of insulin may occur in microorganisms.

INSULIN BINDING TO THE HL-60 HUMAN PROMYELOCYTIC CELL LINE

The HL-60 cell line derived from a patient with promyelocytic leukemia may be grown in liquid suspension cell culture. It binds [125I]-iodoinsulin in a highly specific, rapid, reversible, temperature and pH dependent manner. The pH maximum is approximately 7.8 and binding is maximal after 80 min at 22C with virtually 90% specific. Specific binding is linear with cell number over the range of 107 to 108 cells. The displacement of bound [125I]-iodoinsulin by unlabeled insulin yields curvilinear Scatchard plots at 22C. Using a 2 site model the insulin binding characteristics may best be defined by a “high” affinity site (n1=8.7±2.6(SD)·103 per cell; k1=2.7±0.55·108M-1) and a “low” affinity site (n2=1.12±0.32·105 per cell; k2=7.8±3.6·106M-1). To determine insulin release from its receptor, cells were exposed to [125I]-iodoinsulin for 90 min at 22C. Aliquots were diluted 100-fold in binding buffer or buffer plus 1.67·10-6M insulin. The excess unlabeled insulin accelerated the dissociation of labeled insulin at 22, 30 and 37C, but did not at 4 or 15C. Displacement of [125I]-iodoinsulin by vertebrate insulins and modified porcine insulins is in agreement with their known biological activities. Purified multiplication stimulating factor was less than 1 percent as effective as porcine insulin. These data indicate that the HL-60 cells maintain specific, high affinity insulin receptors. The release of insulin is accelerated by unlabeled hormone above 22C but not below 15C.

Insulin-like and glucagon-like peptides in insect hemolymph

Haemolymph from the tobacco hornworm, Manduca sexta (L.), was examined for insulin-like, glucagon-like and gastrin-like peptides. Several immunoreactive components similar in size to vertebrate insulin and glucagon were separated by gel filtration of acid extracts of larval and pupal haemolymph. No gastrin-like peptide was detected. These results provide evidence that at least two of these peptide hormones are transported in haemolymph to target tissues in insects.

The presence of a hypoglucemic and hypotrehalocemic hormone in the neurosecretory system of the blowfly Calliphora erythrocephala

Extirpation of the median neurosecretory cells (MNC) of the female blowfly, Calliphora erythrocephala, resulted in significant hyperglucemia and hypertrehalocemia after 24 hr and an even greater effect after 48 hr. Both fresh homogenates of the MNC and acid-alcohol extracts of the head, when injected into flies 24 hr after MNC extirpation, caused significant hypoglucemia, and hypotrehalocemia within 4 hr. In normal flies injection of acid-alcohol extract of the head caused a significant hypoglucemia, whereas trehalose values showed a small significant increase. These results suggest the existence of a hormone, localized in the pericarya of the median neurosecretory cells, and having at least one biological similarity to vertebrate insulin.

Insulin-like hypoglycemic and immunological activities in honeybee royal jelly

An aqueous extract of royal jelly from Apis mellifera produced hypoglycemia when injected into larvae of Manduca sexta. Application of specific radioimmunoassay to the partially-purified extract showed that royal jelly contains several insulin-like peptides, the major immunoreactive component of which has an apparent mol. wt similar to that of bovine insulin. These results suggest the existence of a peptide in the honeybee having both biological and structural similarities to vertebrate insulin.

Glucagon-like and insulin-like hormones of the insect neurosecretory system

Aqueous extracts of corpus cardiacum-corpus allatum complexes of the adult tobacco hornworm Manduca sexta produced both glycogenolysis and hypoglycaemia when injected into the larval form of the same species. Application of specific radioimmuno assays to similar extracts showed also that these gland complexes contain both glucagon-like and insulin-like peptides. Further, the partially purified immunoreactive peptides had the expected biological activities. The former decreased the glycogen content of the fatbody and the latter the circulating trehalose levels in recipient animals. These results suggest the existence of hormones in these invertebrates having both biological and structural similarities to vertebrate insulin and glucagon.