~(21)Ala定点突变胰高血糖素及结构与功能变化

 胰高血糖素是由 2 9个氨基酸组成的多肽激素 ,具有促糖元分解的生理功能 ,其拮抗剂有治疗糖尿病病人的潜在应用价值 .在获得重组胰高血糖素基因工程菌基础上 ,利用定点突变技术改造其第 2 1位氨基酸天冬氨酸为丙氨酸 ,并经DNA测序证明胰高血糖素基因发生了点突变 .用IPTG诱导表达后 ,经亲和层析和反相高效液相层析 ,纯化到突变型重组2 1Ala 胰高血糖素 .质谱测定分子量与理论值相符 .利用园二色谱比较重组胰高血糖素和突变的2 1Ala 胰高血糖素在TFE中的二级结构 ,发现胰高血糖素以α螺旋为主要二级结构 ,2 1Ala 胰高血糖素仍有α螺旋结构特征 ,并且含量有所增大 .利用兔升血糖试验 ,发现2 1Ala 胰高血糖素生物活性比重组胰高血糖素减少 51 % (P <0 .0 1 ) .显示天然胰高血糖素第 2 1位氨基酸天冬氨酸与形成α螺旋结构关系不大 ,但在发挥胰高血糖素的生物功能中有重要作用 ,与其可作为钙离子结合位点 ,参与胰高血糖素和受体结合的潜在功能密切相关 .     

Three distinct epitopes on the extracellular face of the glucagon receptor determine specificity for the glucagon amino terminus

The glucagon and glucagon-like peptide-1 (GLP-1) receptors are homologous family B seven-transmembrane (7TM) G protein-coupled receptors, and they selectively recognize the homologous peptide hormones glucagon (29 amino acids) and GLP-1 (30-31 amino acids), respectively. The amino-terminal extracellular domain of the glucagon and GLP-1 receptors (140-150 amino acids) determines specificity for the carboxyl terminus of glucagon and GLP-1, respectively. In addition, the glucagon receptor core domain (7TM helices and connecting loops) strongly determines specificity for the glucagon amino terminus. Only 4 of 15 residues are divergent in the glucagon and GLP-1 amino termini; Ser2, Gln3, Tyr10, and Lys12 in glucagon and the corresponding Ala8, Glu9, Val16, and Ser18 in GLP-1. In this study, individual substitution of these four residues of glucagon with the corresponding residues of GLP-1 decreased the affinity and potency at the glucagon receptor relative to glucagon. Substitution of distinct segments of the glucagon receptor core domain with the corresponding segments of the GLP-1 receptor rescued the affinity and potency of specific glucagon analogs. Site-directed mutagenesis identified the Asp385 --> Glu glucagon receptor mutant that specifically rescued Ala2-glucagon. The results show that three distinct epitopes of the glucagon receptor core domain determine specificity for the N terminus of glucagon. We suggest a glucagon receptor binding model in which the extracellular ends of TM2 and TM7 are close to and determine specificity for Gln3 and Ser2 of glucagon, respectively. Furthermore, the second extracellular loop and/or proximal segments of TM4 and/or TM5 are close to and determine specificity for Lys12 of glucagon.     

Effect of glucagon antiserum on somatostatin,glucagon and insulin release from the isolated perfused rat pancreas

In order to elucidate the effect of glucagon antiserum on the endocrine pancreas, the release of somatostatin, glucagon, and insulin from the isolated perfused rat pancreas was studied following the infusion of arginine both with and without pretreatment by glucagon antiserum. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated both somatostatin and glucagon secretion. However, the responses of somatostatin and glucagon were different at different doses of arginine. The infusion of glucagon antiserum strongly stimulated basal secretion in the perfusate total glucagon (free + antibody bound glucagon) and also enhanced its response to arginine, but free glucagon was undetectable in the perfusate during the infusion. On the other hand, the glucagon antiserum had no significant effect on either insulin or somatostatin secretion. Moreover, electron microscopic study revealed degrannulation and vacuolization in the cytoplasm of the A cells after exposure to glucagon antiserum, suggesting a hypersecretion of glucagon, but no significant change was found in the B cells or the D cells. We conclude that in a single pass perfusion system glucagon antiserum does not affect somatostatin or insulin secretion, although it enhances glucagon secretion.     

Physiological effect of circulating glucagon on the hepatic membrane potential

The pancreatic hormone glucagon hyperpolarizes the liver cell membrane under various conditions. Here we investigated the physiological relevance of this effect by testing the influence of infusions of glucagon antiserum on the liver cell membrane potential in vivo. Intracellular microelectrode recordings of liver cells (up to 60/rat over 2 h) were done in anesthetized male rats. Livers were fixed in place, and recordings were done 10-30 min after intraperitoneal injections of glucagon or hepatic portal vein infusions of glucagon or specific polyclonal glucagon antibodies raised in rabbits. The isotonic lactose vehicle was used as a control for glucagon, and equal amounts of nonimmunized rabbit IgG were used as a control for glucagon antibodies. Intraperitoneal glucagon (400 microg/kg) hyperpolarized the liver cell membrane up to 12 mV, and intraportal glucagon (10 or 60 microg/kg) dose dependently hyperpolarized the liver cell membrane by 3-7 mV. Intraportal infusion of glucagon antiserum (in vitro binding capacity of 4 ng glucagon/rat) significantly depolarized the liver cell membrane by approximately 2.5 mV. The effects of both glucagon and glucagon antiserum reversed after 60-90 min. We conclude that glucagon is a physiologically important modulator of the liver cell membrane potential.     

Role of protein kinase C in the regulation of glucagon gene expression by arginine

The cellular mechanism of glucagon gene expression in intact rat islets and their synthesis and release of glucagon were investigated. Arginine significantly increased the amounts of preproglucagon mRNA and glucagon in the islets and glucagon release. H-7, a specific inhibitor of protein kinase C (PKC), significantly inhibited these effects of arginine. However, H-8, a potent inhibitor of cyclic nucleotide-dependent protein kinases, did not affect the arginine-induced biosynthesis of glucagon or glucagon release. These results suggest that the regulation of glucagon gene expression by arginine is mediated by PKC, not by cyclic nucleotide-dependent protein kinases.     

The purification and biological properties of pancreatic big glucagon.

1. Big glucagon was present in extracts of ox, dog, rat and turkey pancreas, representing 10-15% of the glucagon immunoreactivity, and was shown to be of islet origin by its presence in extracts of isolated pigeon islets. 2. Big glucagon was homogeneous by immunoassay after polyacrylamide-gel electrophoresis and was more electronegative than little glucagon. 3. Big glucagon was purified from bovine pancreas, and its apparent molecular weight was estimated by gel filtration as 8200+/-9%. 4. Limited tryptic proteolysis of the molecule produced an immunoreactive component slightly smaller than little glucagon. 5. Linear dilution curves were obtained with mammalian big glucagons by using both enteroglucagon cross-reacting and 'little-glucagon-carboxyl-end-specific' antisera. 6. The half-times for the disappearance of the immunoreactivity of big and little glucagon that had been injected into the rat circulation were 6.9 and 3.2min respectively. 7. Big glucagon was approximately one-sixth as effective as little glucagon in displacing radioactive little glucagon from its liver membrane receptor. 8. Big glucagon was equipotent on a molar basis with little glucagon in the stimulation of the mouse islet adenylate cyclase, an indicator of insulinogenic activity. 9. On a molar basis, big glucagon inhibited basal liver adenylate cyclase activity to the same extent that little glucagon stimulated the enzyme. 10. Big glucagon was without effect on blood glucose concentration in the rat in doses up to 5mug/kg. 11. Big glucagon was equipotent, on a molar basis, with little glucagon in stimulating lipolysis in isolated chicken fat-cells.     

Adrenergic control of pancreatic glucagon secretion

In order to characterize the adrenergic control of pancreatic A cell, the effect on the glucagon secretion of three sympathomimetic substances (epinephrine, isoproterenol, phenylephrine) and two adrenergic blockers (propranolol and phentolamine) have been separately examined by the isolated perfused rat pancreas. The study was performed in basal state and during glucagon hypersecretion induced by arginine or glucopenia. Epinephrine and isoproterenol infusion determined a prompt an sustained glucagon release both in the basal state and during glucagon hypersecretion. The effect of phenylephrine infusion was slight. In the presence of propranolol, glucagon secretion induced by metabolic stimulus was significantly depressed. The glucagon secretion in the same experimental conditions was insignificantly enhanced by phentolamine. Finally propranolol infusion reverse the glucagon secretion induced by phenylephrine. In conclusion the pancreatic glucagon secretion in our model of study is clearly induced by B adrenergic receptor stimulation.     

Metabolism of glucagon by dipeptidyl peptidase IV (CD26)

Glucagon is a 29-amino acid polypeptide released from pancreatic islet alpha-cells that acts to maintain euglycemia by stimulating hepatic glycogenolysis and gluconeogenesis. Despite its importance, there remains controversy about the mechanisms responsible for glucagon clearance in the body. In the current study, enzymatic metabolism of glucagon was assessed using sensitive mass spectrometric techniques to identify the molecular products. Incubation of glucagon with purified porcine dipeptidyl peptidase IV (DP IV) yielded sequential production of glucagon(3-29) and glucagon(5-29). In human serum, degradation to glucagon(3-29) was rapidly followed by N-terminal cyclization of glucagon, preventing further DP IV-mediated hydrolysis. Bioassay of glucagon, following incubation with purified DP IV or normal rat serum demonstrated a significant loss of hyperglycemic activity, while a similar incubation in DP IV-deficient rat serum did not show any loss of glucagon bioactivity. Degradation, monitored by mass spectrometry and bioassay, was blocked by the specific DP IV inhibitor, isoleucyl thiazolidine. These results identify DP IV as a primary enzyme involved in the degradation and inactivation of glucagon. These findings have important implications for the determination of glucagon levels in human plasma.     

A reassessment of structure-function relationships in glucagon. Glucagon1-21 is a full agonist.

Glucagon1-21 has been prepared by treating native glucagon with carboxypeptidase A. Purified glucagon1-21 did not contain detectable methionine (less than 0.001 residue/mol) and the activity of the compound did not change after treatment with cyanogen bromide as has been shown with native glucagon. Glucagon1-21 stimulates hepatic adenylate cyclase activity to the same extent as native glucagon but with 0.1% the potency. Glucagon1-21 also displayed 0.1% the binding affinity of native glucagon to the glucagon receptor in hepatic membranes. Glucagon22-29 alone or in combination with glucagon1-21 did not activate adenylate cyclase or displase 125I-glucagon from its receptor. The finding that glucagon1-21 is a full agonist on adenylate cyclase is discussed in relation to the structure-function relationships required for the biological action of glucagon.     

Effects of intracerebroventricular injections of des-His1 (Glu9) glucagon amide on the regulatory thermogenesis in muscovy ducklings

Recent investigations have demonstrated a modulatory action of glucagon on shivering via the central nervous system in ducklings. Such an action could be mediated by glucagon receptors that have been recently detected in several brain areas involved in the central control of the involuntary motricity in this avian species. The present study using des-His1 (Glu9) glucagon amide, was performed to investigate the central mechanisms of glucagon on shivering. This glucagon analog was found to be an antagonist of glucagon devoid of adenylate cyclase activity (GR2) by triggering the breakdown of inositol phosphate (GR1) in mammals hepatocytes. The intracerebroventricular administration of des-His1 (Glu9) glucagon amide or glucagon induced a marked inhibition of shivering in ducklings exposed to cold. It seems likely that GR1 receptors contribute to decreased shivering in ducklings exposed to cold. Central glucagon or des-His1 (Glu9) glucagon amide were devoid of thermogenic effect at thermoneutrality.     

Hepatic glucagon clearance during insulin induced hypoglycemia

Studies concerning the importance of glucagon secretion in hypoglycemic counterregulation have assumed that peripheral levels of glucagon are representative of rates of pancreatic glucagon secretion. The measurement of peripheral levels of this hormone, however, may be a poor reflection of secretion rates because of glucagon's metabolism by the liver. Therefore, in order to understand the relationship between pancreatic glucagon secretion and levels of glucagon in the peripheral blood during hypoglycemia, we evaluated hepatic glucagon metabolism during insulin induced hypoglycemia. Four dogs received an insulin infusion to produce glucose levels less than 50 mg/dl for 45 minutes. In response to this, the delivery of glucagon to the liver increased from 36.7 +/- 5.9 ng/min in the baseline to 322.6 +/- 6.3 ng/min during hypoglycemia. Hepatic glucagon uptake increased proportionally from 13.6 +/- 7.2 ng/min to 103.1 +/- 28.3 ng/min and the percentage of delivered hormone that was extracted did not change (30.8 +/- 13.8% vs 32.9 +/- 11.6%). The absolute amount of glucagon metabolized by the liver was dependent on the rate of delivery and was not directly affected by plasma glucose level per se. To directly study the effect of hypoglycemia on hepatic glucagon metabolism, five dogs were given an exogenous infusion of somatostatin followed by an infusion of glucagon and then administered insulin to produce hypoglycemia. The percent of glucagon extracted by the liver (19.5 +/- 4.9% and 21.3 +/- 6.4%) was not affected by a fall in the plasma glucose level.(ABSTRACT TRUNCATED AT 250 WORDS)     

The release and metabolism of pancreatic hormones after major hepatectomy in the dog.

Major hepatectomy in the dog induced a 50% decrease in peripheral serum glucose, a 11-fold increase in portal plasma glucagon and a 36-fold increase in the portal glucagon/insulin ratio 3 hr after operation. Peripheral serum glucose levels were inversely correlated to the logarithmic value of portal plasma glucagon (r = -0.50, p less than 0.01) and that of the portal glucagon/insulin ratio (r = -0.85, p less than 0.01) for 1-6 hr after operation. The ratio of peripheral to portal plasma glucagon was also inversely correlated to the logarithmic value of portal plasma glucagon (r = -0.59, p less than 0.01). In case of glucose infusion, plasma glucagon levels were not elevated after major hepatectomy. The data suggest that glucose deficiency after major hepatectomy in the dog may cause hyperglucagonemia with an enhanced glucagon requirement.     

Partial purification and characterization of the glucagon receptor

Specific labeling of liver plasma membrane glucagon receptors has been achieved by the photoincorporation of a 125I-labeled photoderivative of glucagon, NE-4-azidophenylamidinoglucagon. Identification of glucagon receptors was facilitated by irradiating membranes in the presence of excess unlabeled glucagon. Isoelectric focusing of radioiodinated membrane proteins revealed one major band of glucagon displaceable material which had an isoelectric point of 5.85. When this material was isolated and run on SDS-polyacrylamide gels a major labeled band of Mr55000 was obtained which had properties consistent with those of the glucagon receptor. These studies indicate that a purification of the glucagon receptor of greater than 700-fold can be attained through the use of isoelectric focusing and SDS-polyacrylamide electrophoresis.     

Glucagon-mediated internalization of serine-phosphorylated glucagon receptor and Gsalpha in rat liver

To assess glucagon receptor compartmentalization and signal transduction in liver parenchyma, we have studied the functional relationship between glucagon receptor endocytosis, phosphorylation and coupling to the adenylate cyclase system. Following administration of a saturating dose of glucagon to rats, a rapid internalization of glucagon receptor was observed coincident with its serine phosphorylation both at the plasma membrane and within endosomes. Co-incident with glucagon receptor endocytosis, a massive internalization of both the 45- and 47-kDa Gsalpha proteins was also observed. In contrast, no change in the subcellular distribution of adenylate cyclase or beta-arrestin 1 and 2 was observed. In response to des-His(1)-[Glu(9)]glucagon amide, a glucagon receptor antagonist, the extent and rate of glucagon receptor endocytosis and Gsalpha shift were markedly reduced compared with wild-type glucagon. However, while the glucagon analog exhibited a wild-type affinity for endosomal acidic glucagonase activity and was processed at low pH with similar kinetics and rates, its proteolysis at neutral pH was 3-fold lower. In response to tetraiodoglucagon, a glucagon receptor agonist of enhanced biological potency, glucagon receptor endocytosis and Gsalpha shift were of higher magnitude and of longer duration, and a marked and prolonged activation of adenylate cyclase both at the plasma membrane and in endosomes was observed. The subsequent post-endosomal fate of internalized Gsalpha was evaluated in a cell-free rat liver endosome-lysosome fusion system following glucagon injection. A sustained endo-lysosomal transfer of the two 45- and 47-kDa Gsalpha isoforms was observed. Therefore, these results reveal that within hepatic target cells and consequent to glucagon-mediated internalization of the serine-phosphorylated glucagon receptor and the Gsalpha protein, extended signal transduction may occur in vivo at the locus of the endo-lysosomal apparatus.     

Glucagon-degrading activity in acid-ethanol extract of rat submandibular gland

To investigate whether immunoreactive glucagon really exists in salivary gland, the integrity of glucagon radioimmunoassay was tested in the acid-ethanol extract of rat submandibular gland. Though immunoreactive glucagon was apparently measured in acid-ethanol extract of rat submandibular gland, the extract contained a significant amount of intact glucagon-degrading activity. The apparent % bound in radioimmunoassay highly correlated with the degradation of [125I] glucagon during incubation. Gel filtration profiles of [125I] glucagon incubated with acid-ethanol extract were the same as those of [125I] glucagon damaged by submandibular acid-saline extract. These data suggest that the immunoreactive glucagon in acid-ethanol extract is, as in the case of acid-saline extract, an artifact due to degradation of [125I] glucagon during radioimmunoassay.     

Glucagon antagonists. Synthesis and inhibitory properties of Asp3-containing glucagon analogs

In an effort to find analogs of glucagon that would bind to the glucagon receptor of the rat liver membrane but would not activate membrane-bound adenyl cyclase, several hybrid molecules were synthesized which contained sequences from both glucagon and secretin. [Asp3, Glu9]Glucagon and [Asp3, Glu9, Arg12]glucagon were inactive in the adenyl cyclase assay even at high concentrations but retained some binding affinity for the receptor. They were able to displace 125I-glucagon completely from its receptor and could completely inhibit the activation of adenyl cyclase by natural or synthetic glucagon. The inhibition index [I/A]50 was approximately 110 for both analogs. [Asp3]Glucagon, [Glu3]glucagon and [Asp3, Lys17, 18, Glu21]glucagon were weak partial agonists, while [Asp3, Glu21]glucagon was inactive and a poor inhibitor. The peptides were synthesized by solid-phase methods and purified to homogeneity by reverse-phase high-performance liquid chromatography on C18 silica columns. These are the first fully synthetic competitive glucagon antagonists to be reported.     

Glucagon from avian pancreatic islets: radioreceptor studies.

Glucagon extracted from isolated islets of the pigeon was studied by means of Sephadex gel filtration. Radioreceptor assay, using rat liver plasma membranes and radioiodinated porcine glucagon, showed that the bulk of the activity eluted with glucagon (molecular weight 3500). Avian glucagon appeared to be less effective than porcine glucagon in inhibiting the binding of labeled porcine glucagon to rat plasma membranes.     

Self-association of glucagon as measured by the optical properties of rhodamine 6G

The fluorescence of rhodamine 6G is completely quenched in glucagon solutions in 0.6 M K2HOP4 at pH 10.6. The absorption of rhodamine 6G is red-shifted by the same reaction. A single rhodamine 6G molecule appears to be bound to a hydrophobic patch in the center of the trimer of glucagon. Since the glucagon monomer has almost no organized structure this site exists only in the associated trimer form of glucagon. The self-association of glucagon to the trimer has been determined from the variation in rhodamine 6G fluorescence and absorption measured over a 60-fold range of dye concentration. The self-association constant agrees with values determined by other methods in the absence of dye. The binding isotherms of rhodamine 6G to glucagon shift with glucagon concentration and exhibit negative cooperativity.     

Kinetic studies on hepatic handling of glucagon using the model of non-recirculating perfused rat livers.

Using the model of the in vitro non-recirculating perfused rat liver we studied kinetic aspects of the hepatic handling of glucagon. Under conditions of a 20 min glucagon infusion (glucagon mass flows of 0.05, 0.46 and 4.75 ng/g liver/min, respectively) according to a rectangular profile both total and individual glucagon extractions were dependent on mass flow and time. The time course of glucagon extraction started with an acute phase within the first minute of infusion with a maximum value of 70%, which decreased within the following 30 sec by more than 40%. Depending on concentration, there was a progressive decrease in the hepatic extraction of glucagon up to the end of perfusion. Hepatic glucagon degradation was found to take place only at a little extent. Immediately after terminating the hormone infusion, the liver changed over into a glucagon-releasing organ. Kinetics of glucagon infusion and glucagon-induced hepatic glycogenolysis did not distinguish by parallelism but rather by phase shifting.