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The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are secreted postprandially and contribute importantly to postprandial glucose tolerance. In this study, we assessed the individual and combined contributions of endogenous GIP and GLP-1 to the postprandial changes in glucose and glucoregulatory hormones using the novel GIP receptor antagonist GIP(3-30)NH2 and the well-established GLP-1 receptor antagonist exendin(9-39)NH2. During 4-h oral glucose tolerance tests (75 g) combined with an ad libitum meal test, 18 healthy men received on four separate days in randomized, double-blinded order intravenous infusions of A) GIP(3-30)NH2 (800 pmol/kg/min) plus exendin(9-39)NH2 (0–20 min: 1,000 pmol/kg/min; 20–240 min: 450 pmol/kg/min), B) GIP(3-30)NH2, C) exendin(9-39)NH2, and D) saline, respectively. Glucose excursions were significantly higher during A than during B, C, and D, while glucose excursions during B were higher than during C and D. Insulin secretion (assessed by C-peptide/glucose ratio) was reduced by 37 ± 16% (A), 30 ± 17% (B), and 8.6 ± 16% (C) compared with D (mean ± SD). A and C resulted in higher glucagon levels and faster gastric emptying. In conclusion, endogenous GIP affects postprandial plasma glucose excursions and insulin secretion more than endogenous GLP-1, but the hormones contribute additively to postprandial glucose regulation in healthy individuals.

Tirzepatide (LY3298176) is a dual GIP and GLP-1 receptor agonist under development for the treatment of type 2 diabetes mellitus (T2DM), obesity, and nonalcoholic steatohepatitis. Early phase trials in T2DM indicate that tirzepatide improves clinical outcomes beyond those achieved by a selective GLP-1 receptor agonist. Therefore, we hypothesized that the integrated potency and signaling properties of tirzepatide provide a unique pharmacological profile tailored for improving broad metabolic control. Here, we establish methodology for calculating occupancy of each receptor for clinically efficacious doses of the drug. This analysis reveals a greater degree of engagement of tirzepatide for the GIP receptor than the GLP-1 receptor, corroborating an imbalanced mechanism of action. Pharmacologically, signaling studies demonstrate that tirzepatide mimics the actions of native GIP at the GIP receptor but shows bias at the GLP-1 receptor to favor cAMP generation over β-arrestin recruitment, coincident with a weaker ability to drive GLP-1 receptor internalization compared with GLP-1. Experiments in primary islets reveal β-arrestin1 limits the insulin response to GLP-1, but not GIP or tirzepatide, suggesting that the biased agonism of tirzepatide enhances insulin secretion. Imbalance toward GIP receptor, combined with distinct signaling properties at the GLP-1 receptor, together may account for the promising efficacy of this investigational agent., Studies of the dual GIP and GLP-1 receptor agonist tirzepatide reveal occupancy favoring the GIP receptor and biased cAMP signaling at the GLP-1 receptor.

In patients with type 2 diabetes mellitus (T2DM), the insulinotropic action of the GIP system is desensitized, whereas this is not the case for the GLP‐1 system. This has raised an interesting discussion of whether GIP agonists or antagonists are most suitable for future treatment of T2DM together with GLP‐1‐based therapies. Homozygous carriers of the GIP receptor (GIPR) variant, [E354Q], display lower bone mineral density, increased bone fracture risk and slightly increased blood glucose. Here, we present an in‐depth molecular pharmacological phenotyping of GIPR‐[E354Q]. In silico modelling suggested similar interaction of the endogenous agonist GIP(1‐42) to [E354Q] as to GIPR wt. This was supported by homologous competition binding in COS‐7 cells revealing GIPR wt‐like affinities of GIP(1‐42) with K d values of ~2 nmol/L and wt‐like agonist association rates (K on). In contrast, the dissociation rates (K off) were slower, resulting in 25% higher agonist residence time for GIPR‐[E354Q]. Moreover, in Gαs signalling (cAMP production) GIP(1‐42) was ~2‐fold more potent and more efficacious on GIPR‐[E354Q] compared to wt with 17.5% higher basal activity. No difference from GIPR wt was found in the recruitment of β‐arrestin 2, whereas the agonist‐induced internalization rate was 2.1‐ to 2.3‐fold faster for [E354Q]. Together with the previously described impaired recycling of [E354Q], our findings with enhanced signalling and internalization rate possibly explained by an altered ligand‐binding kinetics will lead to receptor desensitization and down‐regulation. This could explain the long‐term functional impairment of the GIP system in bone metabolism and blood sugar maintenance for [E354Q] carriers and may shed light on the desensitization of the insulinotropic action of GIP in patients with T2DM.

GIP(3-30)NH2 is a high affinity antagonist of the GIP receptor (GIPR) in humans inhibiting insulin secretion via G protein-dependent pathways. However, its ability to inhibit G protein-independent signaling is unknown. Here we determine its action on arrestin-recruitment and receptor internalization in recombinant cells. As GIP is adipogenic, we evaluate the inhibitory actions of GIP(3-30)NH2 in human adipocytes. Finally, we determine the receptor selectivity of GIP(3-30)NH2 among other human and animal GPCRs. cAMP accumulation and β-arrestin 1 and 2 recruitment were studied in transiently transfected HEK293 cells and real-time internalization in transiently transfected HEK293A and in HEK293A β-arrestin 1 and 2 knockout cells. Furthermore, human subcutaneous adipocytes were assessed for cAMP accumulation following ligand stimulation. Competition binding was examined in transiently transfected COS-7 cells using human 125I-GIP(3-30)NH2. The selectivity of human GIP(3-30)NH2 was examined by testing for agonistic and antagonistic properties on 62 human GPCRs. Human GIP(3-30)NH2 inhibited GIP(1-42)-induced cAMP and β-arrestin 1 and 2 recruitment on the human GIPR and Schild plot analysis showed competitive antagonism with a pA2 and Hill slope of 16.8 nM and 1.11 ± 0.02 in cAMP, 10.6 nM and 1.15 ± 0.05 in β-arrestin 1 recruitment, and 10.2 nM and 1.06 ± 0.05 in β-arrestin 2 recruitment. Efficient internalization of the GIPR was dependent on the presence of either β-arrestin 1 or 2. Moreover, GIP(3-30)NH2 inhibited GIP(1-42)-induced internalization in a concentration-dependent manner and notably also inhibited GIP-mediated signaling in human subcutaneous adipocytes. Finally, the antagonist was established as GIPR selective among 62 human GPCRs being species-specific with high affinity binding to the human and non-human primate (Macaca fascicularis) GIPRs, and low affinity binding to the rat and mouse GIPRs (Kd values of 2.0, 2.5, 31.6 and 100 nM, respectively). In conclusion, human GIP(3-30)NH2 is a selective and species-specific GIPR antagonist with broad inhibition of signaling and internalization in transfected cells as well as in human adipocytes.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted postprandially from enteroendocrine K cells, but despite therapeutically interesting effects, GIP physiology in humans remains incompletely understood. Progress in this field could be facilitated by a suitable GIP receptor antagonist. For the first time in humans, we investigated the antagonistic properties of the naturally occurring GIP(3-30)NH2 in in vivo and in in vitro receptor studies. In transiently transfected COS-7 cells, GIP(3-30)NH2 was evaluated with homologous receptor binding and receptor activation (cAMP accumulation) studies at the glucagon-like peptide 1 (GLP-1), glucagon-like peptide-2 (GLP-2), glucagon, secretin and growth hormone-releasing hormone (GHRH) receptors. Ten healthy men (eligibility criteria: age 20–30 years, HbA1c less than 6.5% [48 mmol/mol] and fasting plasma glucose [FPG] less than 7 mmol/l) were included in the clinical study. Data were collected as plasma and serum samples from a cubital vein cannula. As primary outcome, insulin secretion and glucose requirements were evaluated together with in a randomised, four-period, crossover design by infusing GIP(3-30)NH2 (800 pmol kg−1 min−1), GIP (1.5 pmol kg−1 min−1), a combination of these or placebo during hyperglycaemic clamp experiments. The content of the infusions were blinded to the study participants and experimental personnel. No study participants dropped out. GIP(3-30)NH2 neither bound, stimulated nor antagonised a series of related receptors in vitro. The elimination plasma half-life of GIP(3-30)NH2 in humans was 7.6 ± 1.4 min. Markedly larger amounts of glucose were required to maintain the clamp during GIP infusion compared with the other days. GIP-induced insulin secretion was reduced by 82% (p

Enzymatic cleavage of endogenous peptides is a commonly used principle to initiate, modulate and terminate action for instance among cytokines and peptide hormones. The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), and the related hormone glucagon-like peptide-2 (GLP-2) are all rapidly N-terminally truncated with severe loss of intrinsic activity. The most abundant circulating form of full length GIP(1-42) is GIP(3-42) (a dipeptidyl peptidase-4 (DPP-4) product). GIP(1-30)NH2 is another active form resulting from prohormone convertase 2 (PC2) cleavage of proGIP. Like GIP(1-42), GIP (1-30)NH2 is a substrate for DPP-4 generating GIP(3-30)NH2 which, compared to GIP(3-42), binds with higher affinity and very efficiently inhibits GIP receptor (GIPR) activity with no intrinsic activity. Here, we review the action of these four and multiple other N- and C-terminally truncated forms of GIP with an emphasis on molecular pharmacology, i.e. ligand binding, subsequent receptor activation and desensitization. Our overall conclusion is that the N-terminus is essential for receptor activation as GIP N-terminal truncation leads to decreased/lost intrinsic activity and antagonism (similar to GLP-1 and GLP-2), whereas the C-terminal extension of GIP(1-42), as compared to GLP-1, GLP-2 and glucagon (29-33 amino acids), has no apparent impact on the GIPR in vitro, but may play a role for other properties such as stability and tissue distribution. A deeper understanding of the molecular interaction of naturally occurring and designed GIP-based peptides, and their impact in vivo, may contribute to a future therapeutic targeting of the GIP system - either with agonists or with antagonists, or both.

DPP-4 inhibitors, used for treatment of type 2 diabetes, act by increasing the concentrations of intact glucagon-like peptide-1 (GLP-1), but at the same time, they inhibit secretion of GLP-1, perhaps by a negative feedback mechanism. We hypothesized that GLP-1 secretion is feedback regulated by somatostatin (SS) from neighboring D-cells, and blocking this feedback circuit results in increased GLP-1 secretion. We used a wide range of experimental techniques, including gene expression analysis, immunohistochemical approaches, and the perfused mouse intestine to characterize the paracrine circuit controlling GLP-1 and SS. We show that 1) antagonizing the SS receptor (SSTr) 2 and SSTr5 led to increased GLP-1 and SS secretion in the mouse, 2) SS exhibits strong tonic inhibition of GLP-1 secretion preferentially through SSTr5, and 3) the secretion of S was GLP-1 receptor dependent. We conclude that SS is a tonic inhibitor of GLP-1 secretion, and interventions in the somatostain-GLP-1 paracrine loop lead to increased GLP-1 secretion.

Background Glucagon-like peptide-2 (GLP-2) and glucose-dependent insulinotropic polypeptide (GIP) both inhibit bone resorption in humans but the underlying mechanisms are poorly understood. In vitro, GLP-2 activates the GIP-receptor (GIPR). Objective Based on in vitro studies, we hypothesized that the antiresorptive effect of GLP-2 was mediated through the GIPR. This was tested using the selective GIPR-antagonist GIP(3-30)NH2. Methods The study was a randomized, single-blinded, placebo-controlled, crossover study conducted at Hvidovre University Hospital, Denmark. Eight healthy young men were included and studied on four study days: GIP (200 μg), GLP-2 (800 μg), GIP(3-30)NH2 (800 pmol/kg/min) + GLP-2 (800 μg), and placebo. The main outcomes were bone resorption measured as collagen type 1 C-terminal telopeptide (CTX) and bone formation measured as procollagen type 1 N-terminal propeptide (P1NP). Results CTX (mean ± SEM) significantly decreased after both GIP (to 55.3 ± 6.3% of baseline at t = 90 min) and GLP-2 (to 60.5 ± 5.0% of baseline at t = 180 min). The maximal reduction in CTX after GIP(3-30)NH2 + GLP-2 (to 63.2 ± 3.1% of baseline) did not differ from GLP-2 alone (p = 0.95) nor did net AUC0–240 (−6801 ± 879%*min vs −6027 ± 648%*min, p = 0.56). At t = 30 min, GIP significantly (p Conclusions GIPR antagonism did not inhibit the GLP-2-induced reduction in bone resorption (CTX) in healthy young men. In contrast to GLP-2, GIP increased P1NP despite decreasing CTX indicating an uncoupling of bone resorption from formation. Thus, GLP-2 and GIP seem to exert separate effects on bone turnover in humans. Clinical trials information ClinicalTrials.gov ( NCT03159741 ).

Glucose-dependent insulinotropic polypeptide (GIP) is an intestinal hormone with a broad range of physiological actions. In the postprandial state, the hormone stimulates insulin secretion and during eu- and hypoglycemia, it stimulates glucagon secretion. In addition, GIP increases triacylglycerol (TAG) uptake in adipose tissue and decreases bone resorption. However, the importance of these actions in humans are not clearly understood as a specific GIP receptor (GIPR) antagonist - an essential tool to study GIP physiology - has been missing. Several different GIPR antagonists have been identified comprising both peptides, vaccines against GIP, GIP antibodies or antibodies against the GIPR. However, most of these have only been tested in rodents. In vitro, N- and C-terminally truncated GIP variants are potent and efficacious GIPR antagonists. Recently, GIP(3-30)NH2, a naturally occurring peptide, was shown to block the GIPR in humans and decrease GIP-induced insulin secretion as well as adipose tissue blood flow and TAG uptake. So far, there are no studies with a GIPR antagonist in patients with type 2 diabetes (T2D), but because the elevations in fasting plasma glucagon and paradoxical postprandial glucagon excursions, seen in patients with T2D, are aggravated by GIP, a GIPR antagonist could partly alleviate this and possibly improve the fasting and postprandial glycemia. Since the majority of patients with T2D are overweight, inhibition of GIP-induced fat deposition may be beneficial as well. Here we summarize the studies of GIPR antagonists and discuss the therapeutic potential of the GIP system in humans.

Oxyntomodulin is a product of the glucagon precursor, proglucagon, produced and released from the endocrine L-cells of the gut after enzymatic processing by the precursor prohormone convertase 1/3. It corresponds to the proglucagon sequence 33-69 and thus contains the entire glucagon sequence plus a C-terminal octapeptide, comprising in total 37 amino acids. As might have been expected, it has glucagon-like bioactivity, but also and more surprisingly also activates the receptor for GLP-1. This has given the molecule an interesting status as a glucagon-GLP-1 co-agonist, which is currently attracting considerable interest for its potential in the treatment of diabetes and obesity. Here, we provide an update on oxyntomodulin with a focus on its potential role in metabolic diseases.

Summary Glucagon is secreted from pancreatic α cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in β cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion., Graphical Abstract, Highlights • PG 1-61 is a glucagon-like peptide in humans • Hyperglucagonemia may include PG 1-61, as well as glucagon • PG 1-61 stimulates insulin secretion and activates human hepatocytes • PG 1-61 may be a surrogate marker of alpha cell dysfunction, Wewer Albrechtsen et al. identify a glucagon-like molecule, PG 1-61, in humans using mass-spectrometry-based proteomics. PG 1-61 activates the glucagon receptor, stimulates insulin secretion, and activates key gluconeogenic enzymes. In dysmetabolic conditions, PG 1-61 is upregulated and may therefore serve as a marker of alpha cell stress.

Summary Studies on isolated pancreatic islets suggest that neuromedin U (NMU), a brain and gastrointestinal peptide, acts as a decretin hormone, inhibiting glucose-stimulated insulin secretion. We investigated whether this effect could be reproduced in vivo and in isolated perfused rat pancreas. Unlike the incretin hormone, glucagon-like peptide 1 (GLP-1), intravenous NMU administration had no effects on blood glucose and plasma insulin and glucagon in vivo. Moreover, NMU neither changed insulin, glucagon, or somatostatin secretion from isolated perfused rat pancreas, nor affected GLP-1-stimulated insulin and somatostatin secretion. For NMU to act as a decretin hormone, its secretion should increase following glucose ingestion; however, glucose did not affect NMU secretion from isolated perfused rat small intestine, which contained extractable NMU. Furthermore, the two NMU receptors were not detected in endocrine rat or human pancreas. We conclude that NMU does not act as a decretin hormone in rats.