GIP(3-30)NH2 is a potent competitive antagonist of the GIP receptor and effectively inhibits GIP-mediated insulin, glucagon, and somatostatin release.

Alternative processing of the precursor protein pro-GIP results in endogenously produced GIP(1-30)NH2, that by DPP-4 cleavage in vivo results in the metabolite GIP(3-30)NH2. We showed previously that GIP(3-30)NH2 is a high affinity antagonist of the human GIPR in vitro. Here we determine whether it is suitable for studies of GIP physiology in rats since effects of GIP agonists and antagonists are strictly species-dependent. Transiently transfected COS-7 cells were assessed for cAMP accumulation upon ligand stimulation or assayed in competition binding using human 125I-GIP(1-42) as radioligand. In isolated perfused rat pancreata, insulin, glucagon, and somatostatin-releasing properties were evaluated. Competition binding demonstrated that on the rat GIP receptor (GIPR), rat GIP(3-30)NH2 bound with high affinity (Ki of 17nM), in contrast to human GIP(3-30)NH2 (Ki of 250nM). In cAMP studies, rat GIP(3-30)NH2 inhibited GIP(1-42)-induced rat GIPR activation and schild-plot analysis showed competitive antagonism with a pA2 of 13nM and a slope of 0.9±0.09. Alone, rat GIP(3-30)NH2 displayed weak, low-potent partial agonistic properties (EC50>1µM) with an efficacy of 9.4% at 0.32µM compared to GIP(1-42). In perfused rat pancreata, rat GIP(3-30)NH2 efficiently antagonized rat GIP(1-42)-induced insulin, somatostatin, and glucagon secretion. In summary, rat GIP(3-30)NH2 is a high affinity competitive GIPR antagonist and effectively antagonizes GIP-mediated G protein-signaling as well as pancreatic hormone release, while human GIP(3-30)NH2, despite a difference of only one amino acid between the two (arginine in position 18 in rat GIP(3-30)NH2; histidine in human), is unsuitable in the rat system. This underlines the importance of species differences in the GIP system, and the limitations of testing human peptides in rodent systems.

N-terminally and C-terminally truncated forms of glucose-dependent insulinotropic polypeptide are high-affinity competitive antagonists of the human GIP receptor.

BACKGROUND AND PURPOSE: Glucose-dependent insulinotropic polypeptide (GIP) affects lipid, bone and glucose homeostasis. High-affinity ligands for the GIP receptor are needed to elucidate the physiological functions and pharmacological potential of GIP in vivo. GIP(1-30)NH2 is a naturally occurring truncation of GIP(1-42). Here, we have characterized eight N-terminal truncations of human GIP(1-30)NH2 . EXPERIMENTAL APPROACH: COS-7 cells were transiently transfected with human GIP receptors and assessed for cAMP accumulation upon ligand stimulation or competition binding with (125) I-labelled GIP(1-42), GIP(1-30)NH2 , GIP(2-30)NH2 or GIP(3-30)NH2 . KEY RESULTS: GIP(1-30)NH2 displaced (125) I-GIP(1-42) as effectively as GIP(1-42) (Ki 0.75 nM), whereas the eight truncations displayed lower affinities (Ki 2.3-347 nM) with highest affinities for GIP(3-30)NH2 and GIP(5-30)NH2 (5-30)NH2 . Only GIP(1-30)NH2 (Emax 100% of GIP(1-42)) and GIP(2-30)NH2 (Emax 20%) were agonists. GIP(2- to 9-30)NH2 displayed antagonism (IC50 12-450 nM) and Schild plot analyses identified GIP(3-30)NH2 and GIP(5-30)NH2 as competitive antagonists (Ki 15 nM). GIP(3-30) NH2 was a 26-fold more potent antagonist than GIP(3-42). Binding studies with agonist ((125) I-GIP(1-30)NH2 ), partial agonist ((125) I-GIP(2-30)NH2 ) and competitive antagonist ((125) I-GIP(3-30)NH2 ) revealed distinct receptor conformations for these three ligand classes. CONCLUSIONS AND IMPLICATIONS: The N-terminus is crucial for GIP agonist activity. Removal of the C-terminus of the endogenous GIP(3-42) creates another naturally occurring, more potent, antagonist GIP(3-30)NH2 , which like GIP(5-30)NH2 , was a high-affinity competitive antagonist. These peptides may be suitable tools for basic GIP research and future pharmacological interventions.

Species-specific action of (Pro3)GIP - a full agonist at human GIP receptors, but a partial agonist and competitive antagonist at rat and mouse GIP receptors.

BACKGROUND AND PURPOSE: Specific, high potency receptor antagonists are valuable tools when evaluating animal and human physiology. Within the glucose-dependent, insulinotropic polypeptide (GIP) system, considerable attention has been given to the presumed GIP receptor antagonist, (Pro3)GIP, and its effect in murine studies. We conducted a pharmacological analysis of this ligand including interspecies differences between the rodent and human GIP system. EXPERIMENTAL APPROACH: Transiently transfected COS-7 cells were assessed for cAMP accumulation upon ligand stimulation and assayed in competition binding using (125) I-human GIP. Using isolated perfused pancreata both from wild type and GIP receptor-deficient rodents, insulin-releasing, glucagon-releasing and somatostatin-releasing properties in response to species-specific GIP and (Pro3)GIP analogues were evaluated. KEY RESULTS: Human (Pro3)GIP is a full agonist at human GIP receptors with similar efficacy (Emax ) for cAMP production as human GIP, while both rat and mouse(Pro3)GIP were partial agonists on their corresponding receptors. Rodent GIPs are more potent and efficacious at their receptors than human GIP. In perfused pancreata in the presence of 7 mM glucose, both rodent (Pro3)GIP analogues induced modest insulin, glucagon and somatostatin secretion, corresponding to the partial agonist activities observed in cAMP production. CONCLUSIONS AND IMPLICATIONS: When evaluating new compounds, it is important to consider interspecies differences both at the receptor and ligand level. Thus, in rodent models, human GIP is a comparatively weak partial agonist. Human (Pro3)GIP was not an antagonist at human GIP receptors, so there is still a need for a potent antagonist in order to elucidate the physiology of human GIP.

Transfer of liraglutide from blood to cerebrospinal fluid is minimal in patients with type 2 diabetes.

Treatment with liraglutide leads to weight loss. We investigated whether blood-to-cerebrospinal fluid (CSF) transfer of liraglutide occurs, and if so, whether it associates with clinical weight loss following liraglutide treatment in humans. We performed lumbar puncture and blood sampling in eight patients with type 2 diabetes (mean (range)): age 63 (54-79) years; actual body weight: 90 (75-118) kg treated with 1.8 mg liraglutide for 14 (5-22) months and with a treatment-induced weight loss of 8.4 (7-11) kg. We measured liraglutide in plasma and CSF with a radioimmunoassay specific for the N-terminus of the GLP-1 moiety of liraglutide. Mean plasma liraglutide was 31 (range: 21-63) nmol l(-1). The mean CSF-liraglutide concentration was 6.5 (range: 0.9-13.9) pmol l(-1). Ratio of CSF: plasma-liraglutide concentrations was 0.02 (range: 0.07-0.002)% and plasma liraglutide did not correlate with CSF-liraglutide levels (P=0.67). Body weight loss tended to correlate with plasma-liraglutide levels (P=0.06), but not with CSF-liraglutide levels (P=0.69). In conclusion, we measured very low concentrations of liraglutide in CSF, and the levels of CSF liraglutide did not correlate with the actual clinical weight loss in these patients. The amount of liraglutide in plasma tended to correlate with the clinical weight loss.

Gating function of isoleucine-116 in TM-3 (position III:16/3.40) for the activity state of the CC-chemokine receptor 5 (CCR5).

BACKGROUND AND PURPOSE: A conserved amino acid within a protein family indicates a significance of the residue. In the centre of transmembrane helix (TM)-5, position V:13/5.47, an aromatic amino acid is conserved among class A 7TM receptors. However, in 37% of chemokine receptors - a subgroup of 7TM receptors - it is a leucine indicating an altered function. Here, we describe the significance of this position and its possible interaction with TM-3 for CCR5 activity. EXPERIMENTAL APPROACH: The effects of [L203F]-CCR5 in TM-5 (position V:13/5.47), [I116A]-CCR5 in TM-3 (III:16/3.40) and [L203F;G286F]-CCR5 (V:13/5.47;VII:09/7.42) were determined in G-protein- and ß-arrestin-coupled signalling. Computational modelling monitored changes in amino acid conformation. KEY RESULTS: [L203F]-CCR5 increased the basal level of G-protein coupling (20-70% of Emax ) and ß-arrestin recruitment (50% of Emax ) with a threefold increase in agonist potency. In silico, [I116A]-CCR5 switched χ1-angle in [L203F]-CCR5. Furthermore, [I116A]-CCR5 was constitutively active to a similar degree as [L203F]-CCR5. Tyr(244) in TM-6 (VI:09/6.44) moved towards TM-5 in silico, consistent with its previously shown function for CCR5 activation. On [L203F;G286F]-CCR5 the antagonist aplaviroc was converted to a superagonist. CONCLUSIONS AND IMPLICATIONS: The results imply that an aromatic amino acid in the centre of TM-5 controls the level of receptor activity. Furthermore, Ile(116) acts as a gate for the movement of Tyr(244) towards TM-5 in the active state, a mechanism proposed previously for the ß2 -adrenoceptor. The results provide an understanding of chemokine receptor function and thereby information for the development of biased and non-biased antagonists and inverse agonists.