Effects of long-acting analogues of lamprey GLP-1 and paddlefish glucagon on alpha- to beta-cell transdifferentiation in an insulin-deficient transgenic mouse model.

The abilities of the long-acting, dual-agonist anti-diabetic peptides [D-Ala2 ]palmitoyl-lamprey GLP-1 and [D-Ser2 ]palmitoyl-paddlefish glucagon to induce α-cell to ß-cell transdifferentiation were investigated in GluCreERT2 ;ROSA26-eYFP mice. These animals have been genetically engineered so that yellow fluorescent protein is specifically expressed in glucagon-producing α-cells, thereby allowing cell lineage tracing. Insulin deficiency was produced by treatment of the mice with multiple low doses of streptozotocin. Administration of the peptides (twice daily intraperitoneal injections of 25 nmol/kg body weight over 10 days) to streptozotocin-treated mice produced significant (P < 0.05) increases in pancreatic insulin content and plasma insulin concentrations compared with control mice. Immunohistochemical studies demonstrated a significant (P < 0.05) increase in the % of cells staining for both insulin and fluorescent protein in islets located in the head region of the pancreas (from 10.0 ± 1.3% of total cells in untreated mice to 20.0 ± 3.85% in mice treated with D-Ala2 ]palmitoyl-lamprey GLP-1 and to 17.3 ± 1.1% in mice treated with [D-Ser2 ]palmitoyl-paddlefish glucagon). Corresponding effects upon islets in the tail region were not significant. The data indicate an improvement in ß-cell mass and positive effects on transdifferentiation of glucagon-producing to insulin-producing cells. The study provides further evidence that proglucagon-derived peptides from phylogenetical ancient fish show therapeutic potential for treatment of diabetes.

Glucagon from the phylogenetically ancient paddlefish provides a template for the design of a long-acting peptide with effective anti-diabetic and anti-obesity activities.

This study has examined the in vitro and in vivo anti-diabetic properties of the peptidase-resistant analogues [D-Ser2]palmitoyl-paddlefish glucagon and [D-Ser2]palmitoyl-lamprey glucagon. The peptides stimulated insulin release from BRIN-BD11 clonal ß-cells and isolated mouse pancreatic islets and also enhanced cAMP production in cells transfected with the human GLP-1 receptor and with the human glucagon receptor. The insulinotropic actions of the peptides were attenuated in INS-1 cells lacking GLP-1 and glucagon receptors. [D-Ser2]palmitoyl-paddlefish glucagon stimulated proliferation of BRIN-BD11 cells and protected against cytokine-mediated apoptosis as effectively as GLP-1. The analogue was more effective than the native peptide or the lamprey glucagon analogue in acutely lowering blood glucose and elevating plasma insulin in lean mice even when administered up to 4 h before a glucose load. Twice daily administration of [D-Ser2]palmitoyl-paddlefish glucagon to high-fat fed mice over 21 days reduced food intake, body weight, non-fasting blood glucose and plasma insulin concentrations, as well as significantly improving glucose tolerance and insulin resistance and decreasing α-cell area and pancreatic insulin content. Islet expression of the Gcgr, Glp1r, Gipr and Slc2a2 (GLUT-2) genes significantly increased. These data demonstrate that long-acting peptide [D-Ser2]palmitoyl-paddlefish glucagon exerts beneficial metabolic properties in diabetic mice via Ggcr- and Glp1r-activated pathways and so shows potential as a template for further development into an agent for treatment of patients with obesity-related Type 2 diabetes.

A long-acting, dual-agonist analogue of lamprey GLP-1 shows potent insulinotropic, ß-cell protective, and anorexic activities and improves glucose homeostasis in high fat-fed mice.

Peptidase-resistant analogues of GLP-1 peptides from sea lamprey and paddlefish ([D-Ala2]palmitoyl-lamprey GLP-1 and [D-Ala2]palmitoyl-paddlefish GLP-1) produced significant (P ≤ 0.05) and concentration-dependent increases in insulin release from BRIN-BD11 clonal ß-cells and from isolated mouse islets. Both analogues retained the ability of the native peptides to activate both the GLP-1 receptor (GLP1R) and the glucagon receptor (GCGR). [D-Ala2]palmitoyl-lamprey GLP-1 significantly (P < 0.001) stimulated proliferation of BRIN-BD11 cells and protected against cytokine-induced apoptosis. Administration of the lamprey analogue (25 nmol/kg body weight) to lean mice up to 4 h before a glucose load improved glucose tolerance and increased plasma insulin concentrations. Twice daily administration of the lamprey GLP-1 analogue to high fat-fed mice for 21 days decreased body weight, food intake, and circulating glucose and insulin concentrations. The analogue significantly improved glucose tolerance and insulin sensitivity with beneficial effects on islet ß-cell area and insulin secretory responsiveness. Islet gene expression of Glp1r, Gcgr and Gipr significantly increased. The lamprey GLP-1 analogue shows therapeutic promise for treatment of patients with obesity-related Type 2 diabetes.

Glucagon-like peptides-1 from phylogenetically ancient fish show potent anti-diabetic activities by acting as dual GLP1R and GCGR agonists.

Glucagon-like peptides-1 (GLP-1)from phylogenetically ancient fish (lamprey, dogfish, ratfish, paddlefish and bowfin) and from a teleost, the rainbow trout produced concentration-dependent stimulations of insulin release from clonal ß-cells and isolated mouse islets. Lamprey and paddlefish GLP-1 were the most potent and effective. Incubation of BRIN-BD11 cells with GLP-1 receptor (GLP1R) antagonist, exendin-4 (9-39) attenuated insulinotropic activity of all peptides whereas glucagon receptor (GCGR) antagonist [des-His1,Pro4,Glu9] glucagon amide significantly decreased the activities of lamprey and paddlefish GLP-1 only. The GIP receptor antagonist GIP (6-30) Cex-K40 [Pal] attenuated the activity of bowfin GLP-1. All peptides (1 µM) produced significant increases in cAMP concentration in CHL cells transfected with GLP1R but only lamprey and paddlefish GLP-1 stimulated cAMP production in HEK293 cells transfected with GCGR. Intraperitoneal administration of lamprey and paddlefish GLP-1 (25 nmol/kg body weight) in mice produced significant decreases in blood glucose and increased insulin concentrations comparable to the effects of human GLP-1. Lamprey and paddlefish GLP-1 display potent insulinotropic activity in vitro and glucose-lowering activity in vivo that is mediated through GLP1R and GCGR so that these peptides may constitute templates for design of new antidiabetic drugs.

Glucagon-related peptides from phylogenetically ancient fish reveal new approaches to the development of dual GCGR and GLP1R agonists for type 2 diabetes therapy.

The insulinotropic and antihyperglycaemic properties of glucagons from the sea lamprey (Petromyzontiformes), paddlefish (Acipenseriformes) and trout (Teleostei) and oxyntomodulin from dogfish (Elasmobranchii) and ratfish (Holocephali) were compared with those of human glucagon and GLP-1 in mammalian test systems. All fish peptides produced concentration-dependent stimulation of insulin release from BRIN-BD11 rat and 1.1 B4 human clonal ß-cells and isolated mouse islets. Paddlefish glucagon was the most potent and effective peptide. The insulinotropic activity of paddlefish glucagon was significantly (P < 0.01) decreased after incubating BRIN-BD11 cells with the GLP1R antagonist, exendin-4(9-39) and the GCGR antagonist [des-His1,Pro4, Glu9] glucagon amide but GIPR antagonist, GIP(6-30)Cex-K40[palmitate] was without effect. Paddlefish and lamprey glucagons and dogfish oxyntomodulin (10 nmol L-1) produced significant (P < 0.01) increases in cAMP concentration in Chinese hamster lung (CHL) cells transfected with GLP1R and human embryonic kidney (HEK293) cells transfected with GCGR. The insulinotropic activity of paddlefish glucagon was attenuated in CRISPR/Cas9-engineered GLP1R knock-out INS-1 cells but not in GIPR knock-out cells. Intraperitoneal administration of all fish peptides, except ratfish oxyntomodulin, to mice together with a glucose load produced significant (P < 0.05) decreases in plasma glucose concentrations and paddlefish glucagon produced a greater release of insulin compared with GLP-1. Paddlefish glucagon shares the sequences Glu15-Glu16 and Glu24-Trp25-Leu26-Lys27-Asn28-Gly29 with the potent GLP1R agonist, exendin-4 so may be regarded as a naturally occurring, dual-agonist hybrid peptide that may serve as a template design of new drugs for type 2 diabetes therapy.

Evaluation of the insulinotropic and glucose-lowering actions of zebrafish GIP in mammalian systems: Evidence for involvement of the GLP-1 receptor.

The insulinotropic properties of zebrafish GIP (zfGIP) were assessed in vitro using clonal pancreatic ß-cell lines and isolated mouse islets and acute effects on glucose tolerance and insulin release in vivo were evaluated in mice. The peptide produced a dose-dependent increase in the rate of insulin release from BRIN-BD11 rat clonal ß-cells at concentrations ≥30nM. Insulin release from 1.1 B4 human clonal ß-cells and mouse islets was significantly increased by zfGIP (10nM and 1µM). The in vitro insulinotropic activity of zfGIP was decreased after incubating BRIN-BD11 cells with the GLP-1 receptor antagonist, exendin-4(9-39) (p<0.001) and the GIP receptor antagonist, GIP (6-30) Cex-K40[Pal] (p<0.05) but the glucagon receptor antagonist [des-His1,Pro4,Glu9]glucagon amide was without effect. zfGIP (10nM and 1µM) produced significant increases in cAMP concentration in CHL cells transfected with the human GLP-1 receptor but was without effect on HEK293 cells transfected with the human glucagon receptor. Conversely, zfGIP, but not human GIP, significantly stimulated insulin release from CRISPR/Cas9-engineered INS-1 clonal ß-cells from which the GIP receptor had been deleted. Intraperitoneal administration of zfGIP (25 and 75nmol/kg body weight) to mice together with an intraperitoneal glucose load (18mmol/kg body weight) produced a significant decrease in plasma glucose concentrations concomitant with an increase in insulin concentrations. The study provides evidence that the insulinotropic action of zfGIP in mammalian systems involves activation of both the GLP-1 and the GIP receptors but not the glucagon receptor.