Comparative evaluation of the biological activity of glucagon from hens and mammals in in vitro experiments

Crystalline glucagon has been obtained from hens and its biological activity was compared with that of mammalian glucagon. Biological activity of the hormones was evaluated in two test systems; either by hormonal activation of adenylatecyclase in plasmatic membranes of the liver of chicks and rats or by their ability to stimulate lipolysis in chick adipocytes. It was shown that glucagon of hens exhibits its biological activity in the same range of concentrations, as the mammalian hormone (1.10(-9)-1.10(-5) M). Hen glucagon is less effective as compared to mammalian one, irrespectively of the test system used for the assay of its activity.     

Chicken glucagon. Isolation and amino acid sequence studies.

Glucagon was isolated from a side fraction generated during the preparation of insulin and the new pancreatic peptide, avian pancreatic polypeptide from chicken pancreas. The immunological and biological properties are similar to those of beef-pork glucagon. The amino acid composition of chicken glucagon indicates that it contains 1 more serine residue than the porcine hormone and 1 less aspartic acid (asparagine) residue. Thus, chicken glucagon appears to be identical with turkey glucagon.     

Activation of the cAMP cascade by steroidogenic hormones and glucagon in the pathogenic fungus Candida albicans

Incubation of Candida albicans yeast cells with human luteinizing hormone (hLH), human chorionic gonadotrophin (hCG) or glucagon produced a significant rise in cAMP total levels. The effect of these hormones in permeabilized cells of the fungus produced a 2-3 fold increase in the Mg2+, GTP-dependent adenylyl cyclase activity as well as full activation of the cAMP-dependent protein kinase (PKA) activity. These results indicate that the interaction of the mammalian hormones with the fungus triggered the cAMP activation cascade in a similar way to that found in higher eukaryotic organisms.     

Position 9 replacement analogs of glucagon uncouple biological activity and receptor binding.

Recent studies on the glucagon antagonist des-His1-[Glu9]glucagon amide have resulted in pure inhibitors of the hormone, suggesting that the inhibitory properties may be centered around position 9. The present study was designed to investigate the chemical characteristics of substitutions in position 9 of glucagon that determine binding affinity and biological activity. Twenty replacement analogs of position 9 of glucagon were synthesized and assessed for their ability to bind to the glucagon receptor in rat hepatocyte membranes and to activate adenylate cyclase. Any substitution of aspartic acid 9 was accompanied by a severely diminished capacity to transmit the biological signal, while retaining receptor binding affinity. These results are an indication of an uncoupling of receptor binding and biological activity at this locus and define a central role of aspartic acid 9 in glucagon activity. Single replacement or deletion of either His1 or Asp9 in glucagon caused a 20- to 50-fold decrease in cyclase activity, whereas these same changes made in tandem caused virtually complete loss of activity, with decreases of 10(4)-to 10(6)-fold. These observations have led us to speculate that, at the molecular level, the region of glucagon required for transduction of the biological response may be distinct from the binding region and is mediated by a coupled interaction between His1 and Asp9 of the hormone and a complementary functional site of the glucagon receptor.     

A dose-response study of forskolin, stimulatory hormone, and guanosine triphosphate analog on adenylate cyclase from several sources

We have described relationships involving forskolin stimulation of adenylate cyclase (AC) from a variety of sources and the potentiation of forskolin effects by stimulatory hormones (glucagon, ACTH, and epinephrine) and beta, gamma-imidoguanosine 5'-triphosphate (Gpp(NH)p). The effects on AC were examined using membrane preparations of rabbit adipocytes, rat adipocytes, rat erythrocytes, and rat liver. Also examined was the AC of liver membranes of rat pretreated with pertussis toxin as well as that solubilized from rat liver membranes. Maximal forskolin stimulation of AC in all preparations studied revealed a consistent 10-fold increase in AC activity. The EC50 for forskolin was 10 microM for rat liver, 15 microM for rabbit and rat adipocytes and 17 microM for rat erythrocyte AC stimulation. In all cases the AC activity attained by forskolin stimulation was further enhanced by stimulatory hormones in a dose-dependent manner. Furthermore, a combination of all three activators (forskolin, stimulatory hormone, and Gpp(NH)p) resulted in an even greater overall stimulation to levels ranging from 25- to 30-fold over unstimulated activity levels. In the presence of saturating levels of each stimulatory hormone and Gpp(NH)p, the EC50 for forskolin diminished markedly to the range of 0.5 to 4.0 microM. In the absence of any apparent tissue specificity for forskolin stimulation, the general pattern of these results further implicates the catalytic site of the AC complex as the site of forskolin activation. Furthermore, activation of additional components of the complex by Gpp(NH)p and tissue specific hormones may further influence the AC activity and thereby potentiate the stimulation by forskolin.     

Radioreceptor assay for lactogenic hormones based on membranes from rat mammary tumour

A highly sensitive radioreceptor assay (RRA) for human prolactin (hPRL) based on membrane preparations obtained from chemically induced rat mammary tumour is described. The binding of 125I-labelled, highly purified pituitary human prolactin was specific for lactogenic hormones and depending on time, temperature, and concentration of receptor protein. Optimal specific receptor binding (18-20%) was obtained by incubation at 21 degrees C for 18 h. The prolactin receptor was shown to have a single "class" of binding sites with an affinity constant (Ka) of 6.0 X 10(10) mol-1. The binding capacity was 8-33 fmol/mg membrane protein. The sensitivity of the radioreceptor assay was 0.5 ng/ml ovine prolactin (NIH-PS-10) or 0.84 ng/ml human prolactin (NIH-VLS-4). The receptor binding activity of various purified prolactin preparations from different species was comparable to the biological hormone activities, indicating that this in vitro assay system measures values which are biologically relevant.     

Stimulation by prostaglandin E2 of glucagon and insulin release from isolated rat pancreas.

To ascertain whether prostaglandins (PG) may play a role in the secretion of glucagon and in an attempt to elucidate the conflicting observations on the effects of PG on insulin release, the isolated intact rat pancreas was perfused with solutions containing 1.1 x 10(-9) to 1.8 x 10(-5)m PGE2. In the presence of 5.6 mM glucose significant increments in portal venous effluent levels of glucagon and insulin were observed in response to minimal concentrations of 2.8 X 10(-8) and 1.4 X 10(-7) PGE2, respectively; a dose-response relationship was evident for both hormones at higher concentrations of PGE2. When administered over 60 seconds, 1.4 X 10(-6)M PGE2 resulted in a significant increase in glucagon levels within 24 seconds and in insulin within 48 seconds. Ten-minute perfusions of 1.4 X 10(-6)M PGE2 elicited biphasic release of both islet hormones; Phase I glucagon release preceded that of insulin. Both phases of the biphasic glucagon and insulin release which occurred in response to 15-minute perfusions of 10 mM arginine were augmented by PGE2. These observations indicate that PGE2 can evoke glucagon and insulin release at concentrations close to those observed by others in the extracts of rat pancreas. We conclude that PG may be involved in the regulation of secretion of glucagon and insulin and may mediate and/or modify the pancreatic islet hormone response to other secretagogues.     

Application of recombinant DNA technologies to studies on chicken growth hormone

Messenger RNA isolated from chicken pituitaries was used to construct a chicken pituitary cDNA library. A chicken growth hormone cDNA clone was isolated using 32P-labeled mammalian growth hormone cDNA probes. The amino acid sequence (derived from the DNA sequence) of the mature form of chicken growth hormone shows 77% homology with that of bovine growth hormone. The chicken growth hormone cDNA clone was used to generate a vector capable of producing chicken growth hormone in Escherichia coli. The recombinant E. coli-derived chicken growth hormone was similar to pituitary chicken growth hormone in several biochemical and immunological properties. The recombinant-derived hormone has been used to establish a sensitive radioimmunoassay for growth hormone determinations made from chicken sera. The chicken growth hormone gene has also been introduced into a retroviral vector capable of establishing productive infections of chicken cells both in in vitro and in vivo. The resulting infections are accompanied by the production of radioimmunoassay-detectable growth hormone. The concentrations of growth hormone in sera of Leghorn chickens infected with the recombinant retrovirus are three- to tenfold higher than in control animals.     

Nucleotide sequence of the DNA complementary to avian (chicken) preproparathyroid hormone mRNA and the deduced sequence of the hormone precursor

The nucleotide sequence of avian (chicken) prepro-PTH (prepro-PTH) mRNA was determined from a 2.3-kilobase fragment of complementary chicken parathyroid DNA cloned in E. coli MM 924. Northern blot analysis of chicken parathyroid mRNA, using both bovine and chicken cDNA probes, showed that the mRNA (2.3 kilobases) for chicken hormone precursor was approximately 3 times the size of mRNA for mammalian prepro-PTH. Cleavage of the cloned DNA with restriction endonuclease Pstl resulted in three fragments, each of which was subjected to sequence determination. The hormone sequence deduced from the DNA showed that chicken prepro-PTH mRNA encoded a 119-amino acid precursor which included a 25-amino acid signal sequence, a six-residue prohormone peptide, and an 88-amino acid hormone. The hormonal peptide was four residues longer than all known mammalian homologs and included gene deletions and insertions. There was significant homology of sequence in the biologically active 1-34 region with mammalian hormones, but much less in the middle and carboxyl-terminal regions. This is the first nonmammalian PTH sequence to be determined and should prove useful in studying evolution of the gene as well as structure-function relationships of the hormone.     

Opossum insulin, glucagon and pancreatic polypeptide: Amino acid sequences

Pancreatic hormones have been purified from the opossum, a New World marsupial. Opossum insulin contains a Leu substitution at the N-terminus of the B-chain in place of the Phe that is generally present in mammalian insulins. In addition, there are two other amino acid substitutions in the opossum insulin A-chain (positions 8 and 18) compared to pig insulin. Opossum glucagon is identical to chicken glucagon with both differing from the usual mammalian glucagon by Ser in place of Asn at its penultimate C-terminal position. Opossum PP differs from the porcine peptide in only 3 sites (position 3, 19 and 30).     

Gut-islet endocrinology-some evolutionary aspects

Immunological and biological studies have shown that many of the mammalian gastroenteropancreatic (GEP) hormones have counterparts in lower vertebrates. Hormonal localization in cyclostomes and fishes suggests that insulin was phylogenetically the first islet hormone, followed by somatostatin, glucagon and, last, pancreatic polypeptide (PP). Some of the GEP peptides are present in the central and peripheral nervous system of lower vertebrates as well as mammals. GEP hormone-like substances resembling insulin, somatostatin, glucagon, PP, gastrin, secretin, VIP, substance P and enkephalin also occur in protostomian invertebrates (Annelida, Arthropoda, Mollusca), particularly in their nervous system. These findings indicate that the vertebrate hormones may have originated in neural tissue before the development of the vertebrate line of evolution.     

Identification of Gln8-GnRH and His5,Trp7,Tyr8-GnRH in the hypothalamus and extrahypothalamic brain of the ostrich (Struthio camelus)

Gonadotropin-releasing hormone (GnRH) molecular forms were studied in extracts of ostrich hypothalamus and extrahypothalamic brain using high performance liquid chromatography, radioimmunoassay with region-specific antisera and assessment of luteinizing hormone (LH)-releasing activity using chicken dispersed pituitary cells. Two molecular forms of GnRH with chromatographic, immunological and biological properties identical to those of Gln8-GnRH and His5,Trp7,Tyr8-GnRH were demonstrated in both the hypothalamic and extrahypothalamic brain extracts. A greater proportion of His5,Trp7,Tyr8-GnRH was present in the hypothalamus than in extrahypothalamic brain. It is likely that these two forms of GnRH are present in all bird species, since the chicken and the ostrich have evolved separately.     

Identification of His5,Trp7,Tyr8-GnRH (chicken GnRH II) in amphibian brain

GnRH immunoreactive and bioactive peptides in Xenopus laevis brain extract were investigated by high performance liquid chromatography (HPLC), radioimmunoassay with region-specific antisera raised against GnRH (mammalian), His5,Trp7,Tyr8-GnRH (chicken II) and Tyr3,Leu5,Glu6,Trp7,Lys8-GnRH (lamprey), and by assessment of biological activity. Two immunoreactive peptides eluted in the same positions as GnRH and His5,Trp7,Tyr8-GnRH respectively in HPLC systems which were specifically designed to separate four known natural vertebrate GnRHs (mammalian, chicken I and II, salmon). The immunological properties of these two immunoreactive peaks, determined by relative interaction with three region-specific antisera raised against mammalian GnRH and two specific His5,Trp7,Tyr8-GnRH antisera, were identical to those of GnRH and His5,Trp7,Tyr8-GnRH. The immunoreactive peak co-eluting with His5,Trp7,Tyr8-GnRH represented approximately one-third of the total brain GnRH. Both immunoreactive peaks stimulated luteinizing hormone (LH) release in a chicken dispersed pituitary cell bioassay, and the amounts of LH release stimulated by the two peaks were appropriate for these peaks being GnRH and His5,Trp7,Tyr8-GnRH. A small hydrophobic peak with GnRH immunoreactivity eluted in the same position as Trp7,Leu8-GnRH (salmon), while Gln8-GnRH (chicken I) and lamprey GnRH were not detected. Two additional rather hydrophilic peptides cross-reacted with a COOH-terminus-directed antiserum and had LH-releasing activity. LH-releasing activity was also detected in hydrophobic HPLC fractions. In summary, these data provide evidence for the presence of both GnRH and a second peptide with properties identical to His5,Trp7,Tyr8-GnRH in X. laevis brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the binding of human growth hormone to microsomal membranes from rat liver.

The binding of 125I-labelled human growth hormone to the 100000g microsomal membrane fraction prepared from the livers of normal female rats was dependent on time, temperature, pH, membrane concentration and concentration of 125I-labelled human growth hormone. At 22 degrees C binding reached a steady state after 16h, with the mean maximal specific binding being 20% of the tracer initially added. Dissociation of 125I-labelled human growth hormone from the membranes, after addition of excess of unlabelled hormone, was relatively slow with a half-time greater than 24h. Only minor degradation of the 125I-labelled human growth hormone was observed during incubation with membranes for 16 or 25h at 22 degrees C. Similarly, no significant change in the ability of membranes to bind human growth hormone was evident after preincubation of the membranes for 16 or 25h. Specificity studies showed that up to 90% of the 125I-labelled human growth hormone bound could be displaced by 1 mug of unlabelled hormone. Ovine prolactin also showed considerable competition for the binding site. Non-primate growth-hormone preparations (ovine, bovine, porcine and rat) and non-related hormones (insulin, thyrotropin, lutropin and follitropin) all showed negligible competition. Scatchard analysis of the binding data was consistent with two classes of binding site with binding affinities of 0.64 X 10(10) +/- 0.2 X 10(10)M-1 and 0.03 X 10(10) +/- 0.007 X 10(10)M-1 and corresponding binding capacities of 98.4 +/- 10 fmol/mg of protein and 314.6 +/- 46.3 fmol/mg of protein. These studies provide data which, in general, are consistent with the criteria required for hormone-receptor interaction. However, proof of the thesis that the human-growth-hormone-binding sites in female rat liver represent physiological receptors must await the demonstration of a correlation between hormone binding and a biological response.     

Internalization and degradation of glucagon by human mononuclear cells

The processing of glucagon by circulating human mononuclear cells was examined. Glucagon bound to the membrane with a turnover time of 4.4 minutes per site after 15 minutes of incubation and 8 minutes per site after 90 minutes. The amount of intact intracellular hormone increased by 3-fold by 90 minutes suggesting a slowing of intracellular processing with prolonged incubation. Excess unlabelled insulin also slowed the processing of glucagon at the degradative step with no effect on binding or internalization of glucagon. Subcellular fractionation of the cells showed that most hormone accumulated in the 500xg pellet and in the 100,000xg supernatant. N-ethylmaleimide blocked intracellular glucagon degradation suggesting a role for intracellular sulfhydryl-dependent enzymes. Kinetic analysis of the dissociation of glucagon revealed a second order process with K values of 2.2 X 10(-2) fm-1 min-1 and 1.4 X 10(-2) fm-1 min-1 for dissociation from membranes and from membranes + intracellular sites, respectively. T 1/2 values were 6 min. for membrane dissociation and 9 min for membranes + cells. These findings suggest that glucagon interaction with mononuclear cells has characteristics similar to other receptor bound ligands including internalization processing and metabolism.     

Aurintricarboxilic acid as a tool for investigating the template-bound and unbound forms of RNA polymerase I in permeabilized cells

Several polypeptide hormones of apparently diverse structure and function have a number of similarities which suggest that there may be common features in their mechanism of action. These hormones are all composed of a single linear sequence of about 30 amino acids; their hydrophobic amino acids are regularly spaced at every third or fourth amino acid residue, allowing them to form amphipathic structures which can interact with phospholipids; a fragment at or near their N-terminus is required for biological activity. These hormones include glucagon, beta-endorphin, parathyroid hormone and calcitonin. A model is proposed in which all regions of the hormone bind to the receptor with comparable affinity except for a small segment which, when intact, triggers a conformational change in the receptor resulting in a further stabilization of the hormone-receptor complex. The activity of partial sequences and chemically modified forms of beta-endorphin, parathyroid hormone and glucagon are discussed in relation to this model.     

Characterization of prolactin receptors in pig mammary gland.

Prolactin receptors present in the particulate fraction of lactating pig mammary gland were solubilized by 7.5mM-3-[(3-cholamidopropyl)dimethylammonio]-1-propane-su lph onic acid (Chaps) and purified by affinity chromatography on prolactin coupled to Affi-Gel 10. Nearly 30% of the particulate receptors were solubilized by the detergent and over a 1000-fold purification from homogenates was achieved. A water-soluble fraction rich in receptors was observed during the preparation of membranes, although this fraction has not yet been purified. Prolactin binding to the receptors was a time-dependent, reversible and saturable reaction in particulate, Chaps-solubilized and purified receptors. In all forms, receptors showed the same specificity to peptide hormones. Prolactin and human growth hormone bound to the same receptors, whereas bovine growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and insulin failed to bind. After solubilization, the dissociation constant (Kd) for prolactin was decreased 5-fold from 9.8 X 10(-11) M in the particulate receptors to 1.8 X 10(-11) M in solubilized and purified receptors, being due principally to an increase in the association rate constant from 1.0 X 10(9)M-1 X h-1 to (3.9-4.6) X 10(9)M-1 X h-1, respectively, with the dissociation rate constant remaining unchanged at (1.1-1.3) X 10(-2)h-1. Isoelectric focusing of the prolactin-receptor complex revealed two peaks, one at a pI of 5.5-5.6 and the other at 5.2-5.3. Microsomal receptors were covalently cross-linked to 125I-labelled ovine prolactin with ethylene glycol bis(succinimidyl succinate) and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Autoradiography of the gel revealed a major subunit of Mr 28 000-35 000 and a minor one of Mr 67 000-69 000. Anti-(prolactin receptor) antibodies raised against rabbit mammary gland prolactin receptors were equally effective in inhibiting prolactin binding to particulate, solubilized and affinity-purified receptors, suggesting that purified prolactin receptors have a structure indistinguishable immunologically from particulate receptors and rabbit mammary gland prolactin receptors. The present demonstration shows that particulate prolactin receptors from a domestic animal can be solubilized and purified without losing the original properties of high affinity and binding specificity for hormones.     

Significance of charge on lysine residue of ovine luteinizing hormone on immunological and biological properties of the hormone

In order to understand the significance of positive charge of lysine residues of ovine luteinizing hormone (oLH) on immunological and biological activity, the epsilon-NH2 group(s) of ovine LH were sequentially modified with 2-iminothiolane (2IT) that preserves the positive charge of the lysine while the overall charge of the hormone remains unchanged. These studies have also been compared with the oLH modified by N-succinimidyl 3-(2 pyridyldithio) propionate (SPDP) and succinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP) that abolish positive charge of lysine residues. The modification primarily occurs in the alpha-subunit. Sequential modification led to progressive reduction in receptor binding and immunological activities. However, the steroidogenic activity was substantially retained. The immunoreactivity and receptor binding properties of 2IT modified oLH (oLH-2IT) were less affected when compared to SPDP (oLH-SPDP) or LC-SPDP (oLH-LC-SPDP) modified derivatives suggesting that increase in hydrophobic carbon chain in oLH-LC-SPDP molecule resulted in drastic inhibition in immunological and biological properties. But the steroidogenic potential of oLH-2IT, oLH-LC-SPDP or oLH-SPDP was relatively comparable. This suggests that a single -NH2 group modification with 2IT would generate the site in the hormone for conjugation to the toxin/carrier proteins that may retain better immunological and biological activity compared to that of SPDP or LC-SPDP modified oLH.     

Structure-function relationships in glucagon: properties of highly purified des-His-1-, monoiodo-, and (des-Asn-28, Thr-29)(homoserine lactone-27)-glucagon.

We have compared the ability of glucagon and three highly purified derivatives of the hormone to activate hepatic adenylate cyclase (an expression of biological activity of the hormone) and to compete with [125]glucagon for binding to sites specific for glucagon in hepatic plasma membranes. Relative to that of glucagon, biological activity and affinity of [des-Asn-28,Thr-29](homoserine lactone-27)-glucagon, prepared by CNBr treatment of glucagon, were reduced equally by 40- to 50-fold. By contrast, des-His-1-glucagon, prepared by an insoluble Edman reagent and highly purified (less than 0.5% contamination with native glucagon), displayed a 15-fold decrease in affinity but a 50-fold decrease in biological activity relative to that of the native hormone. At maximal stimulating concentrations, des-His-1-glucagon yielded 70% of the activity given by saturating concentrations of glucagon. Thus, des-His-1-glucagon can be classified as a partial weak agonist. Highly purified monoiodoglucagon and native glucagon displayed identical biological activity and affinity for the binding sites. Our findings suggest that the hydrophilic residues at the terminus of the carboxy region of glucagon are involved in the process of recognition at the glucagon receptor but do not participate in the sequence of events leading to activation of adenylate cyclase. The amino-terminal histidyl residue in glucagon plays an important but not obligatory role in the expression of hormone action and contributes to a significant extent in the recognition process.     

Histological analysis of glucagon-like peptide-1 receptor expression in chicken pancreas

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrient ingestion inhibits both gastrointestinal emptying and gastric acid secretion and promotes satiety. The main biological effect of GLP-1 is the stimulation of insulin secretion (thereby fulfilling the criterion for an incretin hormone) in order to reduce blood glucose levels in mammalian species. Chicken GLP-1 receptor (cGLP-1R) has also been identified in various tissues by gene expression analysis. Although certain effects of GLP-1 in mammals and birds are consistent, e.g., inhibition of food intake, whether GLP-1 has the same insulinotropic activity in chickens as in mammals is debated. Moreover, the expression of cGLP-1R in chicken pancreatic B cells has not been reported. The localization of cGLP-1R and its mRNA in pancreatic islets is studied by triple-immunofluorescence microscopy and in situ hybridization. Triple-immunofluorescence microscopy with antisera against cGLP-1R, somatostatin and insulin or glucagon revealed that cGLP-1R protein was exclusively localized in D cells producing somatostatin in chicken pancreatic islets. The D cells were localized in peripheral areas of the pancreatic islets and cGLP-1R mRNA was detected in the same areas, indicating that cGLP-1R mRNA was also expressed in D cells. This is the first report to demonstrate that cGLP-1R is expressed by D cells, not B cells as in mammals. Our study suggests that chicken GLP-1 performs its insulinotropic activity by a different mode of action from that of the mammalian hormone.