干预胰高血糖素信号通路治疗糖尿病的研究进展

胰高血糖素是胰岛素最重要的拮抗激素,其从胰岛α细胞合成后分泌入血,与靶组织的胰高血糖素受体结合,激活靶信号通路,生成环一磷酸腺苷(cAMP),促进糖原分解和糖异生,升高血糖.愈来愈多的研究显示,通过抑制α细胞产生和分泌胰高血糖素、中和血循环胰高血糖素、胰高血糖素受体拮抗剂、抑制胰高糖素受体基因表达等干预胰高血糖素的信号通路的措施有可能成为治疗糖尿病的新方法.     

Design,synthesis, and effects of novel phenylpyrimidines as glucagon receptor antagonists

The hormone glucagon increases blood glucose levels through increasing hepatic glucose output. In diabetic patients, dysregulation of glucagon secretion contributes to hyperglycemia. Thus, the inhibition of glucagon receptor is one target for the treatment of hyperglycemia in type 2 diabetes. Here we designed and synthesized a series of small molecules based on phenylpyrimidine. Of these, the compound (R)-7a most significantly decreased the glucagon-induced cAMP production and glucagon-induced glucose production during in vitro and in vivo assays. In addition, (R)-7a showed good efficacy in glucagon challenge tests and lowered blood glucose levels in diabetic db/db mice. Our results suggest that the compound (R)-7a could be a potential glucose-lowering agent for treating type 2 diabetes.     

Design and synthesis of conformationally constrained tri-substituted ureas as potent antagonists of the human glucagon receptor

A series of conformationally constrained tri-substituted ureas were synthesized, and their potential as glucagon receptor antagonists was evaluated. This effort resulted in the identification of compound 4a, which had a binding IC50 of 4.0 nM and was shown to reduce blood glucose levels at 3 mg/kg in glucagon-challenged mice containing a humanized glucagon receptor. Compound 4a was efficacious in correcting hyperglycemia induced by a high fat diet in transgenic mice at an oral dose as low as 3 mg/kg.     

Discovery of cyclic guanidines as potent, orally active, human glucagon receptor antagonists

In the course of the development of an aminobenzimidazole class of human glucagon receptor (hGCGR) antagonists, a novel class of cyclic guanidine hGCGR antagonists was discovered. Rapid N-dealkylation resulted in poor pharmacokinetic profiles for the benchmark compound in this series. A strategy aimed at blocking oxidative dealkylation led to a series of compounds with improved rodent pharmacokinetic profiles. One compound was orally efficacious in a murine glucagon challenge pharmacodynamic model and also significantly lowered glucose levels in a murine diabetes model.     

Discovery of potent and orally bioavailable indazole-based glucagon receptor antagonists for the treatment of type 2 diabetes

Type 2 diabetes mellitus (T2DM) is characterized by chronically elevated plasma glucose levels. The inhibition of glucagon-induced hepatic glucose output via antagonism of the glucagon receptor (GCGR) using a small-molecule antagonist is a promising mechanism for improving glycemic control in the diabetic state. The present work discloses the discovery of indazole-based β-alanine derivatives as potent GCGR antagonists through an efficient enantioselective synthesis and structure-activity relationship (SAR) exploration and optimization. Compounds within this class exhibited excellent pharmacokinetic properties in multiple preclinical species. In an acute dog glucagon challenge test, compound 13K significantly inhibited glucagon-mediated blood glucose increase when dosed orally at 10 mg/kg.     

A novel series of glucagon receptor antagonists with reduced molecular weight and lipophilicity

A novel series of glucagon receptor antagonists has been discovered. These pyrazole ethers and aminopyrazoles have lower molecular weight and increased polarity such that the molecules fall into better drug-like property space. This work has culminated in compounds 44 and 50 that were shown to have good pharmacokinetic attributes in dog, in contrast to rats, in which clearance was high; and compound 49, which demonstrated a dose-dependent reduction in glucose excursion in a rat glucagon challenge experiment.     

Discovery of novel, potent, selective, and orally active human glucagon receptor antagonists containing a pyrazole core

A novel class of 1,3,5-pyrazoles has been discovered as potent human glucagon receptor antagonists. Notably, compound 26 is orally bioavailable in several preclinical species and shows selectivity towards cardiac ion channels, other family B receptors such hGIP and hGLP1, and a large panel of enzymes and additional receptors. When dosed orally, compound 26 is efficacious in suppressing glucagon induced plasma glucose excursion in rhesus monkey and transgenic murine pharmacodynamic models at 1 and 10 mpk, respectively.     

Human glucagon receptor antagonists based on alkylidene hydrazides

A series of alkylidene hydrazide derivatives containing an alkoxyaryl moiety was optimized. The resulting hydrazide-ethers were competitive antagonists at the human glucagon receptor. Pharmacokinetic experiments showed fast clearance of most of the compounds tested. A representative compound [4-hydroxy-3-cyanobenzoic acid (4-isopropylbenzyloxy-3,5-dimethoxymethylene)hydrazide] with an IC50 value of 20 nM was shown to reduce blood glucose levels in fasted rats.     

Discovery of N-aryl-2-acylindole human glucagon receptor antagonists

A novel class of N-aryl-2-acylindole human glucagon receptor (hGCGR) antagonists is reported. These compounds demonstrate good pharmacokinetic profiles in multiple preclinical species. One compound from this series, indole 33, is orally active in a transgenic murine pharmacodynamic model. Furthermore, a 1mg/kg oral dose of indole 33 lowers ambient glucose levels in an ob/ob/hGCGR transgenic murine diabetes model. This compound was deemed suitable for preclinical safety studies and was found to be well tolerated in an 8-day experimental rodent tolerability study. The combination of preclinical efficacy and safety observed with compound 33 highlights the potential of this class as a treatment for type 2 diabetes.     

Glucagon and regulation of glucose metabolism

As a counterregulatory hormone for insulin, glucagon plays a critical role in maintaining glucose homeostasis in vivo in both animals and humans. To increase blood glucose, glucagon promotes hepatic glucose output by increasing glycogenolysis and gluconeogenesis and by decreasing glycogenesis and glycolysis in a concerted fashion via multiple mechanisms. Compared with healthy subjects, diabetic patients and animals have abnormal secretion of not only insulin but also glucagon. Hyperglucagonemia and altered insulin-to-glucagon ratios play important roles in initiating and maintaining pathological hyperglycemic states. Not surprisingly, glucagon and glucagon receptor have been pursued extensively in recent years as potential targets for the therapeutic treatment of diabetes.     

Somatostatin receptor subtype 5 regulates insulin secretion and glucose homeostasis

Somatostatin (SRIF) regulates pancreatic insulin and glucagon secretion. In the present study we describe the generation of SRIF receptor subtype 5 knockout (sst(5) KO) mice to examine the role of SRIF receptor subtypes (sst) in regulating insulin secretion and glucose homeostasis. Mice deficient in sst(5) were viable, fertile, appeared healthy, and displayed no obvious phenotypic abnormalities. Pancreatic islets isolated from sst(5) KO mice displayed increased total insulin content as compared with islets obtained from wild-type (WT) mice. Somatostatin-28 (SRIF-28) and the sst(5)/sst(1)-selective agonist compound 5/1 potently inhibited glucose-stimulated insulin secretion from WT islets. SRIF-28 inhibited insulin secretion from sst(5) KO islets with 16-fold less potency while the maximal effect of compound 5/1 was markedly diminished when compared with its effects in WT islets. sst(5) KO mice exhibited decreased blood glucose and plasma insulin levels and increased leptin and glucagon concentrations compared with WT mice. Furthermore, sst(5) KO mice displayed decreased susceptibility to high fat diet-induced insulin resistance. The results of these studies suggest sst(5) mediates SRIF inhibition of pancreatic insulin secretion and contributes to the regulation of glucose homeostasis and insulin sensitivity. Our findings suggest a potential beneficial role of sst(5) antagonists for alleviating metabolic abnormalities associated with obesity and insulin resistance.     

Intra-islet insulin suppresses glucagon release via GABA-GABAA receptor system

Excessive secretion of glucagon is a major contributor to the development of diabetic hyperglycemia. Secretion of glucagon is regulated by various nutrients, with glucose being a primary determinant of the rate of alpha cell glucagon secretion. The intra-islet action of insulin is essential to exert the effect of glucose on the alpha cells since, in the absence of insulin, glucose is not able to suppress glucagon release in vivo. However, the precise mechanism by which insulin suppresses glucagon secretion from alpha cells is unknown. In this study, we show that insulin induces activation of GABAA receptors in the alpha cells by receptor translocation via an Akt kinase-dependent pathway. This leads to membrane hyperpolarization in the alpha cells and, ultimately, suppression of glucagon secretion. We propose that defects in this pathway(s) contribute to diabetic hyperglycemia.     

Discovery of furan-2-carbohydrazides as orally active glucagon receptor antagonists

Furan-2-carbohydrazides were found as orally active glucagon receptor antagonists. Starting from the hit compound 5, we successfully determined the structure activity relationships of a series of derivatives obtained by modifying the acidity of the phenol. We identified the ortho-nitrophenol as a good scaffold for glucagon receptor inhibitory activity. Our efforts have led to the discovery of compound 7l as a potent glucagon receptor antagonist with good bioavailability and satisfactory long half-life.     

Glycemic control in mice with targeted disruption of the glucagon receptor gene.

The action of glucagon in the liver is mediated by G-coupled receptors. To examine the role of glucagon in glucose homeostasis, we have generated mice in which the glucagon receptor was inactivated (GR(-/-) mice). Blood glucose levels were somewhat reduced in GR(-/-) mice relative to wild type, in both the fed and fasted state. Plasma insulin levels were not significantly affected. There was no significant effect on fasting plasma cholesterol or triglyceride levels associated with deletion of the glucagon receptor. Glucose tolerance, as assessed by an oral glucose tolerance test, improved. Plasma glucagon levels were strikingly elevated in both fed and fasted animals. Despite a total absence of glucagon receptors, these animals maintained near-normal glycemia and normal lipidemia, in the presence of circulating glucagon concentrations that were elevated by two orders of magnitude.     

The role of salt bridge formation in glucagon: an experimental and theoretical study of glucagon analogs and peptide fragments of glucagon

BACKGROUND: Glucagon is a 29-residue peptide produced in the alpha cells of the pancreas that interacts with hepatic receptors to stimulate glucose production and release, via a cAMP-mediated pathway. Type 2 diabetes patients may have an excess of glucagon and, as such, glucagon antagonists might serve as diabetes drugs. The antagonists that bind to the glucagon receptor but do not exhibit activity could be analogs of glucagon. The presence of salt bridges between some residues of glucagons (such as aspartic acid) and others (such as lysine) might influence both the binding to the receptor and the activity. MATERIALS AND METHODS: Experimental-The solid phase method with 4-methylbenzilhydrilamine resin (p-MBHA resin) was used for the synthesis of glucagon analogs. Rat liver membranes were prepared from male Sprague-Dawley rats by the Neville procedure. The receptor binding essay was performed in 1% BSA, 1 mM dithiothreitol, 25 mM Tris-HCl buffer, pH 7.2. Adenyl cyclase activity was measured in an assay medium containing 1% serum albumin, 25 mM MgCl2, 2 mM dithiothreitol, 0.025 mM GTP, 5 mM ATP, 0.9 mM theophylline, 17.2 mM creatine phosphate, and 1 mg/ml creatine phosphokinase. Theoretical-Quantum chemical calculations using the Titan program with the 6-31G* basis set were performed to calculate the binding energies of salt bridges between aspartic or glutamic acids and lysine. The relative stability of cyclic conformations of glucagon segments versus the extended segments was determined. RESULTS: It was found that the cyclic Glu9-Lys12 amide compound displayed a 20-fold decrease in binding affinity. DesHis1 cyclic compounds Glu20-Lys24 amide and DesHis1Glu9 Glu20-Lys24 amide behave as glucagon antagonists. The calculations show that cyclic conformations of tetrapeptidic and pentapeptidic segments of glucagon are more stable than the extended species. CONCLUSIONS: The biological data and the theoretical calculations show that an intramolecular salt bridge might impart stability to some glucagon antagonists and, when situated at the C-terminus of glucagon, might facilitate induction of an alpha-helix upon initial hormone association with the membrane bilayer. These findings might be a useful tool for the design of new glucagon antagonists.     

Expression of glucagon receptors in tetracycline-inducible HEK293S GnT1- stable cell lines: an approach toward purification of receptor protein for structural studies

Glucagon is a 29-amino acid polypeptide hormone secreted by pancreatic A cells. Together with insulin, it is an important regulator of glucose metabolism. Type 2 diabetes is characterized by reduced insulin secretion from pancreatic B cells and increased glucose output by the liver which has been attributed to abnormally elevated levels of glucagon. The glucagon receptor (GR) is a member of family B G protein-coupled receptors, ligands for which are peptides composed of 30-40 amino acids. The impetus for studying how glucagon interacts with its membrane receptor is to gain insight into the mechanism of glucagon action in normal physiology as well as in diabetes mellitus. The principal approach toward this goal is to design and synthesize antagonists of glucagon that will bind with high affinity to the GR but will not activate it. Site-directed mutagenesis of the GR has provided some insight into the interactions between glucagon and GR. The rational design of potent antagonists has been hampered by the lack of structural information on receptor-bound glucagon. To obtain adequate amounts of receptor protein for structural studies, a tetracycline-inducible HEK293S GnT1(-) cell line that stably expresses human GR at high-levels was developed. The recombinant receptor protein was characterized, solubilized, and isolated by one-step affinity chromatography. This report describes a feasible approach for the preparation of human GR and other family B GPCRs in the quantities required for structural studies.     

Quantitation of glucagon by radioreceptorassay

Receptors for glucagon on rat liver membranes were characterized. They bound [125I] glucagon rapidly in a specific and saturable way. Addition of unlabelled glucagon displaced [125I] glucagon from the binding sites in a concentration dependent way. Concentrations from 10(-9) to 10(-8) M of glucagon caused a linear reduction of binding of labelled glucagon. This concentration interval was used for a three-point assay which fulfilled statistical requirements for validity. Individual assays normally resulted in potency estimates of high precision and statistical weight. Mean values for glucagon activity of preparations tested by receptor assay were within the fiducial limits (P = 0.95) for corresponding activity determined by the rabbit blood glucose method. The receptor assay is less time consuming and requires only part of one rat liver while the in vivo assay uses 16 rabbits. Thus, the receptor assay is less resource demanding and should serve well as a screening instrument for control of potency of glucagon preparations.     

Glucagon-stimulated but not isoproterenol-stimulated glucose formation inhibition by interleukin-6 in primary cultured rat hepatocytes.

During prolonged sepsis, impairment of glucose supply by the liver leads to hypoglycemia. Our aim was to investigate whether proinflammatory cytokine interleukin-6, a major mediator of the hepatic acute phase reaction, could contribute to this impairment by inhibiting hepatic glucose production stimulated by glucagon or isoproterenol in rat hepatocytes. Interleukin-6 inhibited the stimulation of glucose formation from glycogen by glucagon but not by isoproterenol in cultured rat hepatocytes. This was confirmed in the perfused rat liver. In cultured hepatocytes, the increase in cyclic adenosine-3',5'-monophosphate formation by glucagon was inhibited by interleukin-6, which was probably due to attenuation of glucagon binding to the glucagon receptor. The increase in cyclic adenosine-3',5'-monophosphate stimulated by isoproterenol was not affected by interleukin-6. However, the cytokine inhibited both expression of the key gluconeogenic control enzyme, phosphoenolpyruvate carboxykinase, stimulated by glucagon and isoproterenol. Thus, while increased glucose demand during the acute-phase reaction might initially be accomplished by catecholamine-mediated stimulation of glucose formation from glycogen, inhibition of gluconeogenesis by interleukin-6 may contribute to the impairment of glucose homeostasis during the prolonged acute phase reaction.     

A comparative study of several serotonin receptor antagonists on basal and stimulus induced release of insulin and glucagon in the intact rat.

Several commonly used serotonin receptor antagonists were studied for their ability to influence basal plasma insulin and glucagon (using 30K antibody) levels as well as the response of these hormones to a glucose or arginine challenge administered systematically to overnight fasted rats. Cyproheptadine, in contrast to other antagonists employed, induced large increases of insulin, glucagon and glucose, although this hyperinsulinemia was of a smaller magnitude when compared with hormone levels observed during an equivalent hyperglycemia resulting from glucose administration. The pancreatic response to a glucose load (increased insulin and decreased glucagon release) and an arginine load (increased insulin and glucagon release) were prevented by cyproheptadine pretreatment. Basal insulin levels were bot consistently altered by methysergide or cinanserin and were slightly elevated by metergoline. Basal glucagon levels were unaffected by these drugs. These three agents potentiated the insulinotropic effect of an arginine load whereas only metergoline exerted a similar effect on the response to glucose loading. Glucagon release in response to these stimuli was not significantly altered by drug pretreatment.