A novel GIP-oxyntomodulin hybrid peptide acting through GIP, glucagon and GLP-1 receptors exhibits weight reducing and anti-diabetic properties.

Oxyntomodulin (Oxm) is a 37-amino acid peptide linked to alleviation of obesity-diabetes through a dual mode of action mediated at both glucagon and GLP-1 receptors. GIP is the principle physiological regulator of postprandial insulin secretion. Therefore, the primary aim was to design a novel GIP-Oxm peptide incorporating the actions of GIP, GLP-1 and glucagon in a single molecule. The first 11 N-terminal residues of Oxm were substituted with the sequence of stable dA(2)GIP molecule to generate a novel GIP-Oxm peptide (dA(2)GIP-Oxm). dA(2)GIP-Oxm was resistant to DPP-IV and significantly stimulated in vitro insulin release. dA(2)GIP-Oxm stimulated cAMP production in GIP-R, glucagon-R and GLP-1-R transfected cells by up to 95%, 83% and 77% of that elicited by respective native ligands. Acute administration of dA(2)GIP-Oxm to HFF mice resulted in reduced plasma glucose (45% reduction) and increased insulin concentrations (1.7-fold increase). Furthermore, dA(2)GIP-Oxm lowered plasma glucose (42% reduction) and increased plasma insulin (1.6-fold increase) when administered to HFF mice four hours prior to a glucose load. Once-daily administration of dA(2)GIP-Oxm for 15 days in HFF mice lowered body weight (13% reduction), reduced plasma glucose (40% reduction) and increased plasma insulin (1.7-fold increase). Furthermore, glycemic responses were improved (38% reduction) and glucose-mediated plasma insulin concentrations enhanced (2-fold increase). These improvements in metabolic control were independent of changes in food intake and insulin sensitivity. dA(2)GIP-Oxm exerts positive beneficial actions on glucose homeostasis, beta-cell insulin secretion and body weight, mediated through GIP, glucagon and GLP-1 receptors. Such multiple-acting peptides may hold promise as novel therapies for obesity-diabetes.

Administration of an acylated GLP-1 and GIP preparation provides added beneficial glucose-lowering and insulinotropic actions over single incretins in mice with Type 2 diabetes and obesity.

The present study examined the glucose-lowering and insulinotropic properties of acylated GLP-1 (glucagon-like peptide-1) and GIP (glucose-dependent insulinotropic polypeptide) peptides in Type 2 diabetes and obesity. GLP-1, GIP, Liraglutide, N-AcGIP(Lys(37)Myr) (N-acetylGIP with myristic acid conjugated at Lys(37)), a simple combination of both peptides and a Lira-AcGIP preparation [overnight preparation of Liraglutide and N-AcGIP(Lys(37)Myr)] were incubated with DPP-IV (dipeptidyl peptidase-IV) to assess peptide stability, and BRIN-BD11 cells were used to evaluate cAMP production and insulin secretion. Acute glucose-lowering and insulinotropic actions were evaluated in Swiss TO mice. Subchronic studies on glucose homoeostasis, insulin secretion, food intake and bodyweight were evaluated in ob/ob mice. Liraglutide, N-AcGIP(Lys(37)Myr), a simple combination of both peptides and the Lira-AcGIP preparation demonstrated improved DPP-IV resistance (P<0.001), while stimulating cAMP production and insulin secretion (1.4-2-fold; P<0.001). The Lira-AcGIP preparation was more potent at lowering plasma glucose (20-51% reduction; P<0.05-P<0.001) and stimulating insulin secretion (1.5-1.8-fold; P<0.05-P<0.001) compared with Liraglutide and N-AcGIP(Lys(37)Myr) or a simple peptide combination. Daily administration of the Lira-AcGIP preparation to ob/ob mice lowered bodyweight (7-9%; P<0.05), food intake (23%; P<0.05) and plasma glucose (46% reduction; P<0.001), while increasing plasma insulin (1.5-1.6-fold; P<0.001). The Lira-AcGIP preparation enhanced glucose tolerance, insulin response to glucose and insulin content (P<0.05-P<0.001). These findings demonstrate that a combined preparation of the acylated GLP-1 and GIP peptides Liraglutide and N-AcGIP(Lys(37)Myr) markedly improved glucose-lowering and insulinotropic properties in diabetic obesity compared with either incretin mimetic given individually.

Characterization and biological actions of N-terminal truncated forms of glucose-dependent insulinotropic polypeptide.

The N-terminal domain of glucose-dependent insulinotropic polypeptide (GIP) plays an important role in regulating biological activity. This study examined biological properties of several N-terminal truncated forms of GIP and two novel forms with substitutions at Phe position-6 with Arg or Val. GIP(6-42), GIP(R6-42), GIP(V6-42), GIP(7-42) and GIP(9-42) stimulated cAMP production in BRIN-BD11 cells similar to native GIP, whereas responses to GIP(3-42), GIP(4-42), GIP(5-42) and GIP(8-42) were reduced (P<0.01 to P<0.001). GIP-induced cyclic AMP production was significantly inhibited by GIP(3-42), GIP(4-42), GIP(5-42), GIP(6-42), GIP(R6-42), GIP(7-42) and GIP(8-42) (P<0.001). Compared with native GIP, in vitro insulinotropic activity of GIP(3-42), GIP(4-42), GIP(5-42), GIP(7-42) and GIP(8-42) was reduced (P<0.05 to P<0.001), with GIP(4-42), GIP(5-42), GIP(7-42) and GIP(8-42) also potently inhibiting GIP-stimulated insulin secretion (P<0.001). In ob/ob mice, GIP(4-42) and GIP(8-42) increased (P<0.05 to P<0.01) plasma glucose concentrations compared to the glucose-lowering action of native GIP. When GIP(8-42) was co-administered with native GIP it countered the ability of the native peptide to lower plasma glucose and increase circulating insulin concentrations. These data confirm the importance of the N-terminal region of GIP in regulating bioactivity and reveal that sequential truncation of the peptide yields novel GIP receptor antagonists which may have functional significance.

Effects of gamma-glutamyl linker on DPP-IV resistance, duration of action and biological efficacy of acylated glucagon-like peptide-1.

Liraglutide, a GLP-1 mimetic has recently been approved for clinical use in obesity-diabetes. The purpose of this study was to assess if acylation of Liraglutide via its gamma-glutamyl linker contributes to DPP-IV inhibition and efficacy of the molecule, given that such an approach could be useful in prolonging bioactivity of related peptides. Liraglutide lacking the gamma-glutamyl linker (Lira-gammaGlu) and Liraglutide exhibited enhanced DPP-IV resistance with extension of t(1/2) plus effective cAMP production (EC(50): 0.15+/-0.11 and 0.16+/-0.11nM, respectively) compared to GLP-1 (EC(50) 3.81+/-0.80nM). GLP-1, Lira-gammaGlu and Liraglutide increased insulin secretion compared to glucose (1.5-3.0-fold; p<0.05 to p<0.001). In vivo, Lira-gammaGlu and Liraglutide significantly lowered plasma glucose when administered 4 and 8h prior to a glucose load (1.3-1.9-fold; p<0.05 to p<0.001). Twice-daily administration of Lira-gammaGlu and Liraglutide for 14 days significantly decreased food intake (1.2-fold; p<0.05) and plasma glucose (1.1-1.6-fold; p<0.05 to p<0.01) whilst increasing plasma insulin (1.4-1.6-fold; p<0.05). At 14 days, Lira-gammaGlu and Liraglutide markedly improved glucose tolerance (1.4-3.4-fold; p<0.05 to p<0.001), insulin response to glucose (1.4-1.5-fold; p<0.05), insulin sensitivity (1.3-1.4-fold; p<0.05 to p<0.01), as well as increasing pancreatic insulin content (1.4-fold; p<0.05). Functional characteristics of Lira-gammaGlu and Liraglutide are almost indistinguishable, questioning necessity of gamma-glutamyl linker in acylation for generation of long-acting incretin mimetics.

Fatty acid derivatised analogues of glucose-dependent insulinotropic polypeptide with improved antihyperglycaemic and insulinotropic properties.

C-terminal acylation of Lys(37) with myristic (MYR; tetradecanoic acid), palmitic (PAL; hexadecanoic acid) and stearic (octadecanoic acid) fatty acids with or without N-terminal acetylation was employed to develop long-acting analogues of the glucoregulatory hormone, glucose-dependent insulinotropic polypeptide (GIP). All GIP analogues exhibited resistance to dipeptidylpeptidase-IV (DPP-IV) and significantly improved in vitro cAMP production and insulin secretion. Administration of GIP analogues to ob/ob mice significantly lowered plasma glucose-GIP(Lys(37)MYR), N-AcGIP(Lys(37)MYR) and GIP(Lys(37)PAL) increased plasma insulin concentrations. GIP(Lys(37)MYR) and N-AcGIP(Lys(37)MYR) elicited protracted glucose-lowering effects when administered 24h prior to an intraperitoneal glucose load. Daily administration of GIP(Lys(37)MYR) and N-AcGIP(Lys(37)MYR) to ob/ob mice for 24 days decreased glucose and significantly improved plasma insulin, glucose tolerance and beta-cell glucose responsiveness. Insulin sensitivity, pancreatic insulin content and triglyceride levels were not changed. These data demonstrate that C-terminal acylation particularly with myristic acid provides a class of stable, longer-acting forms of GIP for further evaluation in diabetes therapy.

Prolonged GIP receptor activation using stable mini-PEGylated GIP improves glucose homeostasis and beta-cell function in age-related glucose intolerance.

In older populations there is significant increase in incidence of impaired glucose tolerance and diabetes. Glucose-dependent insulinotropic polypeptide (GIP) improves glycemic control but its use as a therapeutic is hindered by short biological half-life. The present study examined effects of a longer-acting form of GIP, GIP[mPEG], on glucose homeostasis and beta-cell function in mice with age-related glucose intolerance. GIP[mPEG] decreased glucose and increased insulin concentrations when administered prior to a glucose challenge. Daily administration of GIP[mPEG] for 20 days had no effect on body weight and food intake. However, non-fasting glucose concentrations were decreased and insulin concentrations increased. Glycemic response to intraperitoneal glucose was improved and glucose-mediated insulin secretion enhanced. Insulin sensitivity, circulating triglycerides and resistin levels were unchanged by the treatment regimen, but plasma adiponectin levels increased. These data indicate that prolonged activation of the GIP receptor with GIP[mPEG] counters aspects of impaired beta-cell function and age-related glucose intolerance.

C-terminal mini-PEGylation of glucose-dependent insulinotropic polypeptide exhibits metabolic stability and improved glucose homeostasis in dietary-induced diabetes.

Glucose-dependent insulinotropic polypeptide has been proposed as a potential therapeutic for type 2 diabetes, however, efforts to bring forward this drug have been hindered due to its short circulating half-life. We have adopted a novel strategy to increase potency and prolong GIP action through C-terminal mini-PEGylation (GIP[mPEG]). In contrast to GIP, GIP[mPEG] was resistant to dipeptidylpeptidase-IV (DPP-IV) up to and including 24h. Both GIP[mPEG] and GIP concentration-dependently stimulated cAMP production (EC50 6.6 and 0.7 nM, respectively) and insulin secretion (p < 0.01 to p < 0.001) in pancreatic BRIN-BD11 cells. Acute injection of GIP[mPEG] together with glucose to high fat fed mice significantly lowered plasma glucose (p < 0.05) and increased plasma insulin responses (p < 0.05). Furthermore, GIP[mPEG] markedly lowered plasma glucose when administered 4-24h prior to a glucose load (p < 0.05). Daily administration of GIP[mPEG] for 20 days in high fat mice did not alter body weight, food intake or non-fasting plasma insulin, however, non-fasting plasma glucose concentrations were significantly lowered (p < 0.05). Moreover, glucose tolerance was significantly improved (p < 0.05) together with glucose-mediated plasma insulin responses (p < 0.05). Insulin sensitivity, pancreatic insulin content, triglyceride and adiponectin levels were not changed. In summary, these data demonstrate that C-terminal mini-PEGylation of GIP is a useful strategy to prolong metabolic stability and improve biological action thus representing a novel therapeutic option for type 2 diabetes.